Kit and method for measuring measurement target substance in biological sample

ABSTRACT

An object of the present invention is to provide a kit and a method which are capable of measuring a measurement target substance in a biological sample with high precision in a measurement range from a low concentration range to a high concentration range. According to the present invention, there is provided a kit for measuring a measurement target substance in a biological sample, including a labeled particle having a first binding substance capable of binding to a measurement target substance in a biological sample, and a substrate having a second binding substance capable of binding to any one of the measurement target substance or the first binding substance, in which the labeled particle is a luminescent labeled particle containing at least one kind of compound represented by Formula (1) and a particle. 
     
       
         
         
             
             
         
       
     
     Each symbol in Formula (1) has the meaning described in the present specification.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of PCT International Application No.PCT/JP2018/013406 filed on Mar. 29, 2018, which claims priority under 35U.S.C § 119(a) to Japanese Patent Application No. 2017-066921 filed onMar. 30, 2017. Each of the above application(s) is hereby expresslyincorporated by reference, in its entirety, into the presentapplication.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to a kit for measuring a measurementtarget substance in a biological sample, and a method for measuring ameasurement target substance in a biological sample.

2. Description of the Related Art

A fluorescence detection method is widely used as a highly sensitive andeasy measurement method for quantifying a protein, an enzyme, aninorganic compound, or the like. The fluorescence detection method is amethod for confirming the presence of a measurement target substance bydetecting the fluorescence emitted in the case where excitation light ofa specific wavelength is applied to a sample considered to contain ameasurement target substance which is excited by the light of a specificwavelength to emit fluorescence. In the case where the measurementtarget substance is not a phosphor, for example, the presence of themeasurement target substance can be confirmed by bring a substance inwhich a substance specifically binding to the measurement targetsubstance is labeled with a fluorescent dye into contact with a sample,and then detecting the fluorescence emitted in the case where excitationlight is applied in the same manner as described above.

In the fluorescence detection method as described above, there is knowna method for utilizing the effect of electric field enhancement byplasmon resonance to improve sensitivity for detecting a measurementtarget substance present in a small amount. In this method, in order togenerate plasmon resonance, a sensor chip having a metal layer in apredetermined area on a transparent support is prepared, and excitationlight is incident from a surface side of the support opposite to asurface on which metal layer is formed, with respect to an interfacebetween the support and the metal film, at a predetermined angle equalto or more than the total reflection angle. The surface plasmon isgenerated in the metal layer by the irradiation with the excitationlight, and the signal/noise ratio (S/N ratio) is improved byfluorescence enhancement, which is induced by the electric fieldenhancement effect caused by generation of the surface plasmon, and thushigh-sensitive measurement can be achieved. The fluorescence detectionmethod by surface plasmon excitation (hereinafter referred to as “SPFmethod”) is about 10 times stronger in a signal enhancement degree thanthe fluorescence detection method by epi-excitation (also referred to asepi-fluorescence method), and thus high-sensitive measurement can beachieved.

JP3442777B discloses fluorescent microparticles produced by blending aninitial donor dye having a preferable excitation peak and a finalreceptor dye having a preferable luminescence peak in polymermicroparticles. In JP3442777B, it is described that a polyazaindacenedye is used as the dye.

Olivier Galangau et al., Org. Biomol. Chem., 2010, Vol. 8, pp. 4546 to4553 discloses that a novel distyryl BODIPY® (registered trademark,abbreviation of boron-dipyrromethene) dye is designed and synthesized,and the synthesized distyryl BODIPY® dye has been analyzed forabsorption and emission spectra in a chloromethane solution.

SUMMARY OF THE INVENTION

As described above, although the SPF method is known as a method capableof high-sensitive measurement by a simple measurement method, the SPFmethod is not sufficiently satisfactory for the measurement of a verysmall amount of a measurement target substance. Among the detectionmethods, in a competition method for measuring small molecules thatcannot be sandwiched by antibodies, it has been necessary to reduce theamount of particle in the reaction system in order to raise thedetection sensitivity, but in such a case, there has remained a problemthat measurement in a high concentration range cannot be achieved withhigh precision due to the lack of fluorescence intensity. Thefluorescent microparticles described in JP3442777B have a preferableeffective Stokes shift, but have a problem of low quantum yield. InOlivier Galangau et al., Org. Biomol. Chem., 2010, Vol. 8, pp. 4546 to4553, absorption and emission spectra of a dye solution are analyzed,but there is no description about incorporation of a dye into particles.

The object of the present invention is to provide a kit and a methodcapable of achieving high-precision measurement of a measurement targetsubstance in a biological sample in a wide concentration range from alow concentration to a high concentration.

As a result of intensive studies to achieve the above object, thepresent inventors have found that, in a kit including a labeled particlehaving a first binding substance capable of binding to a measurementtarget substance and a substrate having a second binding substancecapable of binding to any one of the measurement target substance or thefirst binding substance, by using a labeled particle having an emissionmaximum wavelength in a long wavelength range of 680 nm or longer andexhibiting a high quantum yield as the labeled particle, the object canbe achieved. The present invention has been completed based on thesefindings. That is, according to the present invention, the followinginventions are provided.

<1> A kit for measuring a measurement target substance in a biologicalsample, comprising: a labeled particle having a first binding substancecapable of binding to a measurement target substance in a biologicalsample; and a substrate having a second binding substance capable ofbinding to any one of the measurement target substance or the firstbinding substance, in which the labeled particle is a luminescentlabeled particle containing at least one kind of compound represented byFormula (1) and a particle.

In the formula, R¹¹ to R¹⁵ each independently represent a hydrogen atom,a halogen atom, an alkyl group, an aryl group, a heterocyclic group, anethenyl group, an ethynyl group, an amino group, an acyl group, analkoxy group, an aryloxy group, an alkylthio group, or an arylthiogroup, each of which may have a substituent, and at least three of R¹¹,. . . , or R¹⁵ represent atoms or groups other than hydrogen atoms. X¹and X² each independently represent a halogen atom, an alkyl group, anaryl group, a heterocyclic group, a hydroxy group, an alkoxy group, anaryloxy group, an alkylthio group, an arylthio group, an ethenyl group,or an ethynyl group, each of which may have a substituent, and X¹ and X²may be linked to each other to form a ring. Ar¹ and Ar² eachindependently represent an aryl group or a heterocyclic group, each ofwhich may have a substituent. L¹ and L² each independently represent anyone of Formulae (L-1) to (L-4).

In the formulae, R¹¹¹ to R¹¹⁶ each independently represent a hydrogenatom, a halogen atom, an alkyl group, an aryl group, a heterocyclicgroup, an ethenyl group, an ethynyl group, an amino group, an acylgroup, an alkoxy group, an aryloxy group, an alkylthio group, or anarylthio group, each of which may have a substituent. A represents —O—,—S—, or —NH—.

<2> The kit according to <1>, in which the labeled particle is a labeledlatex particle.

<3> The kit according to <1> or <2>, in which the labeled particle has acarboxyl group.

<4> The kit according to any one of <1> to <3>, in which an averageparticle size of the labeled particle is 70 to 500 nm.

<5> The kit according to any one of <1> to <4>, in which the compoundrepresented by Formula (1) is a compound represented by Formula (3).

In the formula, R¹¹, R¹², R¹⁴, R¹⁵, X¹, X², Ar¹, Ar², L¹, and L² are asdefined in Formula (1), provided that at least two of R¹¹, R¹², R¹⁴, orR¹⁵ are atoms or groups other than hydrogen atoms. R³¹ to R³⁵ eachindependently represent a hydrogen atom, a halogen atom, an alkyl group,an aryl group, a heterocyclic group, an ethenyl group, an ethynyl group,an amino group, an acyl group, a cyano group, an alkoxy group, anaryloxy group, an alkylthio group, or an arylthio group, each of whichmay have a substituent, and any one of R³¹, R³², R³⁴, or R³⁵ is a groupconsisting of two or more atoms.

<6> The kit according to any one of <1> to <4>, in which the compoundrepresented by Formula (1) is a compound represented by Formula (4).

In the formula, R¹², R¹³, R¹⁴, X¹, X², Ar¹, Ar², L¹, and L² are asdefined in Formula (1), provided that at least one of R¹², R¹³, or R¹⁴is an atom or group other than a hydrogen atom. R⁴¹ and R⁴² eachindependently represent an aryl group, a heterocyclic group, an ethenylgroup, or an ethynyl group, each of which may have a substituent.

<7> The kit according to any one of <1> to <4>, in which the compoundrepresented by Formula (1) is a compound represented by Formula (5).

In the formula, R¹¹ to R¹⁵, X¹, X², L¹, and L² are as defined in Formula(1). R⁵¹ and R⁵² each independently represent an alkyl group, an arylgroup, a heteroaryl group, an amino group, an acyl group, an alkoxygroup, an aryloxy group, an alkylthio group, or an arylthio group, eachof which may have a substituent. Q¹ and Q² each independently representan aromatic hydrocarbon ring or an aromatic heterocyclic ring, each ofwhich may have a substituent.

<8> The kit according to any one of <1> to <7>, in which the labeledparticle is a luminescent particle containing at least one kind ofenergy donor compound, at least one kind of energy acceptor compound,and a particle, and at least one kind of the energy donor compound orthe energy acceptor compound is the compound represented by Formula (1).

<9> The kit according to <8>, in which at least one kind of compoundrepresented by Formula (1) is contained as the energy donor compound,and at least one kind of compound represented by Formula (1) iscontained as the energy acceptor compound.

<10> The kit according to <8> or <9>, in which a molar ratio of theenergy donor compound to the energy acceptor compound is 1:10 to 10:1.

<11> The kit according to any one of <8> to <10>, in which a Stokesshift between the energy donor compound and the energy acceptor compoundis 40 nm or more.

<12> The kit according to any one of <1> to <11>, in which the substrateincludes a detection area having the second binding substance.

<13> The kit according to <12>, in which the detection area is a metalfilm containing gold.

<14> A method for measuring a measurement target substance in abiological sample, the method comprising: a reaction step of reacting abiological sample with a labeled particle having a first bindingsubstance capable of binding to a measurement target substance; acapturing step of capturing the labeled particle on a substrate having asecond binding substance capable of binding to any one of themeasurement target substance or the first binding substance by bringinga reaction product obtained in the reaction step into contact with thesubstrate; and a label information acquisition step of acquiring labelinformation related to the measurement target substance, in which thelabeled particle is a luminescent labeled particle containing at leastone kind of compound represented by Formula (1) and a particle.

In the formula, R¹¹ to R¹⁵ each independently represent a hydrogen atom,a halogen atom, an alkyl group, an aryl group, a heterocyclic group, anethenyl group, an ethynyl group, an amino group, an acyl group, analkoxy group, an aryloxy group, an alkylthio group, or an arylthiogroup, each of which may have a substituent, and at least three of R¹¹,. . . , or R¹⁵ represent atoms or groups other than hydrogen atoms. X¹and X² each independently represent a halogen atom, an alkyl group, anaryl group, a heterocyclic group, a hydroxy group, an alkoxy group, anaryloxy group, an alkylthio group, an arylthio group, an ethenyl group,or an ethynyl group, each of which may have a substituent, and X¹ and X²may be linked to each other to form a ring. Ar¹ and Ar² eachindependently represent an aryl group or a heterocyclic group, each ofwhich may have a substituent. L¹ and L² each independently represent anyone of Formulae (L-1) to (L-4).

In the formulae, R¹¹¹ to R¹¹⁶ each independently represent a hydrogenatom, a halogen atom, an alkyl group, an aryl group, a heterocyclicgroup, an ethenyl group, an ethynyl group, an amino group, an acylgroup, an alkoxy group, an aryloxy group, an alkylthio group, or anarylthio group, each of which may have a substituent. A represents —O—,—S—, or —NH—.

<15> The method according to <14>, in which the particle is a latexparticle.

<16> The method according to <14> or <15>, in which the particle has acarboxyl group.

<17> The method according to any one of <14> to <16>, in which anaverage particle size of the labeled particle is 70 to 500 nm.

<18> The method according to any one of <14> to <17>, in which thecompound represented by Formula (1) is a compound represented by Formula(3).

In the formula, R¹¹, R¹², R¹⁴, R¹⁵, X¹, X², Ar¹, Ar², L¹, and L² are asdefined in Formula (1), provided that at least two of R¹¹, R¹², R¹⁴, orR¹⁵ are atoms or groups other than hydrogen atoms. R³¹ to R³⁵ eachindependently represent a hydrogen atom, a halogen atom, an alkyl group,an aryl group, a heterocyclic group, an ethenyl group, an ethynyl group,an amino group, an acyl group, a cyano group, an alkoxy group, anaryloxy group, an alkylthio group, or an arylthio group, each of whichmay have a substituent, and any one of R³¹, R³², R³⁴, or R³⁵ is a groupconsisting of two or more atoms.

<19> The method according to any one of <14> to <17>, in which thecompound represented by Formula (1) is a compound represented by Formula(4).

In the formula, R¹², R¹³, R¹⁴, X¹, X², Ar¹, Ar², L¹, and L² are asdefined in Formula (1), provided that at least one of R¹², R¹³, or R¹⁴is an atom or group other than a hydrogen atom. R⁴¹ and R⁴² eachindependently represent an aryl group, a heterocyclic group, an ethenylgroup, or an ethynyl group, each of which may have a substituent.

<20> The method according to any one of <14> to <17>, in which thecompound represented by Formula (1) is a compound represented by Formula(5).

In the formula, R¹¹ to R¹⁵, X¹, X², L¹, and L² are as defined in Formula(1). R⁵¹ and R⁵² each independently represent an alkyl group, an arylgroup, a heteroaryl group, an amino group, an acyl group, an alkoxygroup, an aryloxy group, an alkylthio group, or an arylthio group, eachof which may have a substituent. Q¹ and Q² each independently representan aromatic hydrocarbon ring or an aromatic heterocyclic ring, each ofwhich may have a substituent.

<21> The method according to any one of <14> to <20>, in which thelabeled particle is a luminescent particle containing at least one kindof energy donor compound, at least one kind of energy acceptor compound,and a particle, and at least one kind of the energy donor compound orthe energy acceptor compound is the compound represented by Formula (1).

<22> The method according to <21>, in which at least one kind ofcompound represented by Formula (1) is contained as the energy donorcompound, and at least one kind of compound represented by Formula (1)is contained as the energy acceptor compound.

<23> The method according to <21> or <22>, in which a molar ratio of theenergy donor compound to the energy acceptor compound is 1:10 to 10:1.

<24> The method according to any one of <21> to <23>, in which a Stokesshift between the energy donor compound and the energy acceptor compoundis 40 nm or more.

<25> The method according to any one of <14> to <24>, in which thesubstrate includes a detection area having the second binding substance.

<26> The method according to <25>, in which the detection area is ametal film containing gold.

<27> The method according to <26>, in which label information related tothe measurement target substance is acquired by fluorescence detectiondue to surface plasmon excitation.

According to the kit and the method of the present invention, it ispossible to achieve high-precision measurement of a measurement targetsubstance in a biological sample in a wide concentration range from alow concentration to a high concentration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a 400 MHz ¹H NMR spectrum of Compound (4).

FIG. 2 shows a 400 MHz ¹H NMR spectrum of Compound (7).

FIG. 3 shows a schematic view of a sensor chip.

FIG. 4 shows an exploded view of the sensor chip.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, embodiments of the present invention will be described indetail. In the present specification, the numerical range indicated byusing “to” means a range including numerical values described before andafter “to” as a minimum value and a maximum value, respectively.

[Kit for Measuring Measurement Target Substance in Biological Sample]

A kit for measuring a measurement target substance in a biologicalsample according to the embodiment of the present invention includes alabeled particle having a first binding substance capable of binding toa measurement target substance in a biological sample, and a substratehaving a second binding substance capable of binding to any one of themeasurement target substance or the first binding substance, in whichthe labeled particle is a luminescent labeled particle containing atleast one kind of compound represented by Formula (1) and a particle.

(Biological Sample)

The biological sample is not particularly limited as long as the sampleis a sample that may contain the measurement target substance. Forexample, biologic samples, particularly body fluids (for example, blood,serums, plasma, spinal fluid, tears, sweat, urine, pus, runny nose, orsputum) or excrements (for example, feces), organs, tissues, mucousmembranes, skin, or the like of animals (for example, humans, dogs,cats, horses, or the like) can be mentioned.

(Measurement Target Substance)

The measurement target substance is not particularly limited. Forexample, thyroxine (T4), triiodothyronine (T3), estradiol (E2),aldosterone, symmetrical dimethyl arginine (SDMA), bile acid, cortisol,cholesterol, corticosterone, progesterone, testosterone, estrogen,vitamins, creatinine, amino acids, β-carotene, creatinine, digoxin,theophylline, folic acid, proteins such as inflammatory markers andsepsis markers, or the like can be mentioned.

Progesterone is a sex hormone that is secreted from ovaries and placentaand is involved in luteal function and pregnancy. Progesterone is usedto diagnose menstrual cycle abnormality and infertility. Progesterone isalso used to check the mating timing of dogs and ovarian remnants ofcats.

(First Binding Substance)

The first binding substance used in the present invention is a substancecapable of binding to the measurement target substance. As the firstbinding substance, an antigen, an antibody, or a complex thereof can beused, but the first binding substance is not limited thereto.Preferably, the first binding substance is an antibody. In the casewhere the first binding substance is an antibody, as antibodies capableof binding to the measurement target substance, for example, anantiserum prepared from a serum of an animal immunized with themeasurement target substance, an immunoglobulin fraction purified fromthe antiserum, a monoclonal antibody obtained by cell fusion usingspleen cells of an animal immunized with the measurement targetsubstance, or a fragment thereof [for example, F(ab′)₂, Fab, Fab′, orFv] can be used. Preparation of these antibodies can be performed by aconventional method. Furthermore, the antibody may be modified as in thecase of a chimeric antibody or the like, or a commercially availableantibody or an antibody prepared from an animal serum or culturesupernatant by known methods can be used.

For example, in the case where the measurement target substance isprogesterone, an anti-progesterone antibody capable of binding toprogesterone (preferably, specifically recognizing progesterone) is usedas the first binding substance.

A method for preparing an anti-progesterone antibody is described belowas an example.

A progesterone-BSA conjugate can be prepared by mixing progesterone,bovine serum albumin (hereinafter referred to as BSA), and a condensingagent. Using the conjugate as a mouse immunization antigen, mice aresubcutaneously immunized at the back several times. In this case,complete Freund's adjuvant (CFA) and/or incomplete Freund's adjuvant(IFA) can be appropriately selected and then used as a mixture with theimmunization antigen. The complete Freund's adjuvant is a substance thatstimulates immunity and is a mixture of paraffin and ARLACEL. Theincomplete Freund's adjuvant is an adjuvant in which dead mycobacteriaor dead bacteria of Mycobacterium tuberculosis are added to the completeFreund's adjuvant to further enhance the antigenicity. After severalimmunizations are performed as appropriate for several weeks, a bloodsample is collected from the mice and antibody titers are measured. Theantigen is administered intraperitoneally in the case where a sufficientrise in the antibody titers is observed, and the spleen is isolatedseveral days later. By fusing the spleen cells isolated from theimmunized mice with mutant myeloma cells (myeloma), it is possible toprepare fused cells having an antibody-producing ability. Onlyantibody-producing cells against a target antigen are selected from thefused cells, and limiting dilution is performed to proliferate only thecell line. Culture (cloning) of the cells after dilution can beperformed. The fusion cell line thus obtained is injected into theabdominal cavity of a mouse, monoclonal antibodies can be produced inascites fluid by proliferating ascites-type antibody-producing cells,and thus a target antibody can be obtained by recovering theseantibodies.

(Labeled particle)

The labeled particle used in the present invention is a luminescentlabeled particle containing at least one kind of compound represented byFormula (1) and a particle, and is also referred to as a fluorescentlabeled particle.

The meaning of each symbol in Formula (1) is as defined in the presentspecification.

It is known that an ordinary dye compound is influenced by associationin the case where the amount of incorporation into particles isincreased, and thus the quantum yield decreases (this is also referredto as concentration quenching). In particular, in the case of beingincorporated into particles, a fluorescent dye compound having a longabsorption wavelength of 650 nm or longer tends to exhibit concentrationquenching, whereby it is difficult to maintain a quantum yield.

Inclusion of a conjugated substituent in the compound represented byFormula (1) used in the present invention makes it possible to emitlight of long wavelength and inclusion of a plurality of substituents inthe dipyrromethene skeleton makes it also possible to suppress adecrease in the quantum yield in the polymer particle. As a factor ofsuppressing a decrease in the quantum yield, suppression ofintermolecular interaction (for example, it-n interaction) by aplurality of substituents projecting in a direction perpendicular to thedipyrromethene skeleton is considered. According to the compoundrepresented by Formula (1), it is possible to produce a luminescentlabeled particle (preferably a fluorescent particle, and more preferablya fluorescent nanoparticle) having high luminance, particularly in thelong wavelength range. In the case where the labeled particle is afluorescent particle, the luminance is the fluorescence intensity.According to the present invention, since the luminescence quantum yieldis high in the region of the window of the living body (in the vicinityof 650 to 900 nm which is a near-infrared wavelength range which is easyto transmit through the living body), the sensitivity of sensing usingluminescence can be improved.

In the present specification, the alkyl group may be any of linear,branched, cyclic, or a combination thereof, and the number of carbonatoms in the linear or branched alkyl group is preferably 1 to 36, morepreferably 1 to 18, still more preferably 1 to 12, and particularlypreferably 1 to 6. The cyclic alkyl group may be, for example, acycloalkyl group having 3 to 8 carbon atoms. Specific examples of thealkyl group include a methyl group, an ethyl group, an n-propyl group,an isopropyl group, an n-butyl group, an iso-butyl group, a sec-butylgroup, a t-butyl group, an n-pentyl group, an n-hexyl group, an n-heptylgroup, an n-octyl group, an n-nonyl group, an n-decyl group, ann-undecyl group, an n-dodecyl group, an n-tridecyl group, ann-tetradecyl group, an n-pentadecyl group, an n-hexadecyl group, ann-heptadecyl group, an n-octadecyl group, and a cyclohexyl group.

In the present specification, the aryl group is preferably an aryl grouphaving 6 to 48 carbon atoms, more preferably an aryl group having 6 to24 carbon atoms, and still more preferably an aryl group having 6 to 14carbon atoms, and examples thereof include a phenyl group, a naphthylgroup, an anthryl group, a pyrenyl group, a phenanthrenyl group, abiphenyl group, and a fluorenyl group.

In the present specification, the heterocyclic group is preferably anyof 5- to 7-membered substituted or unsubstituted, saturated orunsaturated, aromatic or non-aromatic, or monocyclic or fusedheterocyclic groups. The heterocyclic group is preferably a heterocyclicgroup having a ring-constituting atom selected from a carbon atom, anitrogen atom, an oxygen atom, and a sulfur atom and having at least onehetero atom selected from a nitrogen atom, an oxygen atom, or a sulfuratom, and more preferably a 5- or 6-membered aromatic heterocyclic grouphaving 3 to 30 carbon atoms. Examples of the heterocyclic group includea furyl group, a benzofuryl group, a dibenzofuryl group, a thienylgroup, a benzothienyl group, a dibenzothienyl group, a pyridyl group, apyrimidinyl group, a quinolyl group, an isoquinolyl group, an acridinylgroup, a phenanthridinyl group, a pteridinyl group, a pyrazinyl group, aquinoxalinyl group, a pyrimidinyl group, a quinazolyl group, apyridazinyl group, a cinnolinyl group, a phthalazinyl group, a triazinylgroup, an oxazolyl group, a benzoxazolyl group, a thiazolyl group, abenzothiazolyl group, an imidazolyl group, a benzimidazolyl group, apyrazolyl group, an indazolyl group, an isoxazolyl group, abenzisoxazolyl group, an isothiazolyl group, a benzisothiazolyl group,an oxadiazolyl group, a thiadiazolyl group, a triazolyl group, atetrazolyl group, a furyl group, a thienyl group, a pyrrolyl group, anindolyl group, an imidazopyridinyl group, and a carbazolyl group.

In the present specification, the acyl group is preferably a linear orbranched alkanoyl group having 2 to 15 carbon atoms, and examplesthereof include an acetyl group, a propionyl group, a butyryl group, anisobutyryl group, a valeryl group, an isovaleryl group, a pivaloylgroup, a hexanoyl group, a heptanoyl group, and a benzoyl group.

In the present specification, the alkoxy group is preferably an alkoxygroup having 1 to 20 carbon atoms, and examples thereof include amethoxy group, an ethoxy group, a propoxy group, an n-butoxy group, apentyloxy group, a hexyloxy group, and a heptyloxy group.

In the present specification, the aryloxy group is preferably an aryloxygroup having 6 to 14 carbon atoms, and examples thereof include aphenoxy group, a naphthoxy group, and an anthryloxy group.

The alkylthio group is preferably an alkylthio group having 1 to 30carbon atoms, and examples thereof include a methylthio group, anethylthio group, and an n-hexadecylthio group.

The arylthio group is preferably an arylthio group having 6 to 30 carbonatoms, and examples thereof include a phenylthio group, ap-chlorophenylthio group, and an m-methoxyphenylthio group.

In the present specification, examples of the halogen atom include afluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

In the present specification, examples of the aromatic ring includearomatic hydrocarbon rings such as a benzene ring, a naphthalene ring,an anthracene ring, a phenanthrene ring, a pyrene ring, a perylene ring,and a terylene ring; aromatic heterocyclic rings such as an indene ring,an azulene ring, a pyridine ring, a pyrazine ring, a pyrimidine ring, apyrazole ring, a pyrazolidine ring, a thiazolidine ring, an oxazolidinering, a pyran ring, a chromene ring, a pyrrole ring, a pyrrolidine ring,a benzimidazole ring, an imidazoline ring, an imidazolidine ring, animidazole ring, a pyrazole ring, a triazole ring, a triazine ring, adiazole ring, an indoline ring, a thiophene ring, a thienothiophenering, a furan ring, an oxazole ring, an oxadiazole ring, a thiazinering, a thiazole ring, an indole ring, a benzothiazole ring, abenzothiadiazole ring, a naphthothiazole ring, a benzoxazole ring, anaphthoxazole ring, an indolenine ring, a benzindolenine ring, apyrazine ring, a quinoline ring, and a quinazoline ring; and fusedaromatic rings such as a fluorene ring and a carbazole ring; among whicharomatic rings having 5 to 16 carbon atoms (aromatic rings and fusedrings containing aromatic rings) are preferable.

In addition, the aromatic ring may have a substituent, and the term“aromatic ring” means both an aromatic ring having a substituent and anaromatic ring having no substituent. As the substituent of the aromaticring, the substituents described in Substituent group A to be mentionedlater can be mentioned.

In the present specification, examples of the amino group include anamino group; an alkyl-substituted amino group such as a mono- ordimethylamino group, a mono- or diethylamino group, or a mono ordi(n-propyl)amino group; an amino group substituted with an aromaticresidue such as a mono- or diphenylamino group or a mono- or adinaphthylamino group; an amino group substituted with one alkyl groupand one aromatic residue, such as a monoalkylmonophenylamino group; abenzylamino group, an acetylamino group, and a phenylacetylamino group.Here, the aromatic residue means a group in which one hydrogen atom isremoved from an aromatic ring, and the aromatic ring is as describedabove in the present specification.

The alkyl group, aryl group, heterocyclic group, ethenyl group, ethynylgroup, amino group, acyl group, alkoxy group, aryloxy group, alkylthiogroup, or arylthio group represented by R¹¹ to R¹⁵ may have asubstituent. Examples of the substituent include the substituentsdescribed in Substituent group A below.

Substituent Group A:

a sulfamoyl group, a cyano group, an isocyano group, a thiocyanatogroup, an isothiocyanato group, a nitro group, a nitrosyl group, ahalogen atom, a hydroxy group, an amino group, a mercapto group, anamido group, an alkoxy group, an aryloxy group, an alkylthio group, anarylthio group, a carbamoyl group, an acyl group, an aldehyde group, acarbonyl group, an aryl group, an alkyl group, an alkyl groupsubstituted with a halogen atom, an ethenyl group, an ethynyl group, asilyl group, and a trialkylsilyl group (such as a trimethylsilyl group).

The alkyl group, aryl group, heterocyclic group, hydroxy group, alkoxygroup, aryloxy group, alkylthio group, arylthio group, ethenyl group, orethynyl group represented by X¹ and X² may have a substituent. Examplesof the substituent include the substituents described in Substituentgroup A.

The aryl group or heterocyclic group represented by Ar¹ and Ar² may havea substituent. Examples of the substituent include the substituentsdescribed in Substituent group A.

The alkyl group, aryl group, heterocyclic group, ethenyl group, ethynylgroup, amino group, acyl group, alkoxy group, aryloxy group, alkylthiogroup, or arylthio group represented by R¹¹¹ to R¹¹⁶ may have asubstituent. Examples of the substituent include the substituentsdescribed in Substituent group A.

<Compound Represented by Formula (1)>

In Formula (1), R¹¹ to R¹⁵ each independently represent a hydrogen atom,a halogen atom, an alkyl group, an aryl group, a heterocyclic group, anethenyl group, an ethynyl group, an amino group, an acyl group, analkoxy group, an aryloxy group, an alkylthio group, or an arylthiogroup, each of which may have a substituent. At least three of R¹¹, . .. , or R¹⁵ represent atoms or groups other than hydrogen atoms,preferably at least four of R¹¹, . . . , or R¹⁵ represent atoms orgroups other than hydrogen atoms, and more preferably all of R¹¹ to R¹⁵represent atoms or groups other than hydrogen atoms.

R¹¹ and R¹⁵ may be the same or different atoms or groups, but arepreferably the same atoms or groups. R¹² and R¹⁴ may be the same ordifferent atoms or groups, but are preferably the same atoms or groups.

R¹¹ and R¹⁵ preferably represent a hydrogen atom, a halogen atom, analkyl group, an aryl group, a heterocyclic group, an ethenyl group or anethynyl group, each of which may have a substituent.

R¹² and R¹⁴ preferably represent an alkyl group which may have asubstituent.

R¹³ preferably represents an aryl group which may have a substituent.

In Formula (1), X¹ and X² each independently represent a halogen atom,an alkyl group, an aryl group, a heterocyclic group, a hydroxy group, analkoxy group, an aryloxy group, an alkylthio group, an arylthio group,an ethenyl group, or an ethynyl group, each of which may have asubstituent, and X¹ and X² may be linked to each other to form a ring.

X¹ and X² preferably represent a halogen atom or an alkoxy group. X¹ andX² are more preferably a fluorine atom, a methoxy group, an ethoxygroup, an isopropyloxy group, or a t-butyloxy group, each of which isalso preferably substituted with a fluorine atom or an alkoxy group.

In Formula (1), Ar¹ and Ar² each independently represent an aryl groupor a heterocyclic group, each of which may have a substituent.

In Formula (1), L¹ and L² each independently represent any one ofFormulae (L-1) to (L-4).

In the formulae, R¹¹ to R¹⁶ each independently represent a hydrogenatom, a halogen atom, an alkyl group, an aryl group, a heterocyclicgroup, an ethenyl group, an ethynyl group, an amino group, an acylgroup, an alkoxy group, an aryloxy group, an alkylthio group, or anarylthio group, each of which may have a substituent. A represents —O—,—S—, or —NH—.

L¹ and L² preferably represent any one of Formula (L-1) or Formula(L-2).

R¹¹¹ to R¹¹⁶ are preferably hydrogen atoms.

<As to Compound Represented by Formula (2)>

A preferred example of the compound represented by Formula (1) is acompound represented by Formula (2).

In the formula, R¹¹ to R¹⁵, X¹, X², Ar¹, and Ar² are as defined inFormula (1), and the preferred ranges thereof are also the same as thepreferred ranges in Formula (1). L²¹ and L²² each independentlyrepresent a group represented by Formula (L-1) or Formula (L-2).

<As to Compound Represented by Formula (3)>

A preferred example of the compound represented by Formula (1) is acompound represented by Formula (3).

In Formula (3), R¹¹, R¹², R¹⁴, R¹⁵, X¹, X², Ar¹, Ar², L¹, and L² are asdefined in Formula (1), and preferred ranges thereof are also the sameas the preferred ranges in Formula (1). Provided that at least two ofR¹¹, R¹², R¹⁴, or R¹⁵ are atoms or groups other than hydrogen atoms,preferably at least three of R¹¹, R¹², R¹⁴, or R¹⁵ are atoms or groupsother than hydrogen atoms, and more preferably R¹¹, R¹², R¹⁴, and R¹⁵are atoms or groups other than hydrogen atoms.

In Formula (3), R³¹ to R³⁵ each independently represent a hydrogen atom,a halogen atom, an alkyl group, an aryl group, a heterocyclic group, anethenyl group, an ethynyl group, an amino group, a cyano group, an acylgroup, an alkoxy group, an aryloxy group, an alkylthio group, or anarylthio group, each of which may have a substituent (examples of thesubstituent include the substituents described in Substituent group A),and any one of R³¹, R³², R³⁴, or R³⁵ is a group consisting of two ormore atoms. The group consisting of two or more atoms is preferably analkyl group, an aryl group, an ethenyl group, an ethynyl group, an aminogroup, a cyano group, or an alkoxy group and more preferably an alkylgroup. Among the alkyl groups, an alkyl group consisting only of carbonatoms and hydrogen atoms or an alkyl group substituted with a halogenatom is preferable; an alkyl group consisting only of 1 to 6 carbonatoms and hydrogen atoms or an alkyl group substituted with a fluorineatom is more preferable; a methyl group, an isopropyl group, a t-butylgroup, or a trifluoromethyl group is still more preferable; and a methylgroup is particularly preferable.

<As to Compound Represented by Formula (4)>

A preferred example of the compound represented by Formula (1) is acompound represented by Formula (4).

In Formula (4), R¹², R¹³, R¹⁴, X¹, X², Ar¹, Ar², L¹, and L² are asdefined in Formula (1), and the preferred ranges thereof are also thesame as the preferred ranges in Formula (1). Provided that at least oneof R¹², R¹³, or R¹⁴ is an atom or group other than a hydrogen atom,preferably at least two of R¹², R¹³, or R¹⁴ are atoms or groups otherthan hydrogen atoms, and more preferably R¹², R¹³, and R¹⁴ are atoms orgroups other than hydrogen atoms.

In Formula (4), R⁴¹ and R⁴² each independently represent an aryl group,a heterocyclic group, an ethenyl group, or an ethynyl group, each ofwhich may have a substituent. Examples of the substituent include thesubstituents described in Substituent group A. R⁴¹ and R⁴² are eachindependently preferably an aryl group, an ethenyl group, or an ethynylgroup, from the viewpoint of improving a quantum yield, an aryl group ispreferable, and from the viewpoint of increasing a wavelength, anethenyl group or an ethynyl group is preferable. In the case of being anaryl group, it is preferred to have at least one substituent at theortho or meta position of the aryl group, and it is more preferred tohave at least one substituent at the ortho position of the aryl group.The number of the substituent substituting for the aryl group ispreferably 1 to 3 and more preferably 2 or 3. The substituentsubstituting for the aryl group is preferably an alkyl group, morepreferably a methyl group, an isopropyl group, or a t-butyl group, andstill more preferably a methyl group.

<As to Compound Represented by Formula (5)>

A preferred example of the compound represented by Formula (1) is acompound represented by Formula (5).

In Formula (5), R¹¹ to R¹⁵, X¹, X², L¹, and L² are as defined in Formula(1), and the preferred ranges thereof are also the same as the preferredranges in Formula (1).

In Formula (5), R⁵¹ and R⁵² each independently represent an alkyl group,an aryl group, a heteroaryl group, an amino group, an acyl group, analkoxy group, an aryloxy group, an alkylthio group, or an arylthiogroup, each of which may have a substituent. Examples of the substituentinclude the substituents described in Substituent group A. R⁵¹ and R⁵²each independently are preferably an alkyl group or an alkoxy group, andfrom the viewpoint of improving a quantum yield, more preferably analkyl group, still more preferably a methyl group, an ethyl group, anisopropyl group, or a t-butyl group, and particularly preferably amethyl group. From the viewpoint of increasing a wavelength, R⁵¹ and R⁵²each independently are more preferably an alkoxy group, still morepreferably a methoxy group, an ethoxy group, an isopropyloxy group, or at-butyloxy group, and particularly preferably a methoxy group.

Q¹ and Q² each independently represent an aromatic hydrocarbon ring oran aromatic heterocyclic ring, each of which may have a substituent.Examples of the substituent include the substituents described inSubstituent group A. Q¹ and Q² are each preferably an aromatichydrocarbon ring, more preferably a benzene ring, a naphthalene ring, ananthracene ring, a phenanthrene ring, or a pyrene ring, still morepreferably a benzene ring or a naphthalene ring, and particularlypreferably a benzene ring. As the group containing R⁵¹ and forming Q¹and the group containing R⁵² and forming Q¹, a tolyl group, a xylylgroup, or a mesityl group is preferable; a xylyl group or a mesitylgroup is more preferable; a xylyl group having methyl groups at bothends of the ortho position relative to the bonding position with L¹ orL², or a mesityl group having methyl groups at both ends of the orthoposition and at the para position relative to the bonding position withL¹ or L² is still more preferable; and a mesityl group having methylgroups at both ends of the ortho position and at the para positionrelative to the bonding position with L¹ or L² is particularlypreferable.

<As to Compound Represented by Formula (6)>

The compound represented by Formula (5) is more preferably a compoundrepresented by Formula (6).

In the formula, R¹¹, R¹², R¹⁴, and R^(s5) each independently represent ahydrogen atom, a halogen atom, an alkyl group, an aryl group, aheterocyclic group, an ethenyl group, an ethynyl group, an amino group,an acyl group, an alkoxy group, an aryloxy group, an alkylthio group, oran arylthio group, each of which may have a substituent, and at leasttwo of R¹¹, R¹², R¹⁴, or R¹⁵ are atoms or groups other than hydrogenatoms. X¹ and X² each independently represent a halogen atom, an alkylgroup, an aryl group, a heterocyclic group, a hydroxy group, an alkoxygroup, an aryloxy group, an alkylthio group, an arylthio group, anethenyl group, or an ethynyl group, each of which may have asubstituent, and X¹ and X² may be linked to each other to form a ring.R³¹ to R³⁵ each independently represent a hydrogen atom, a halogen atom,an alkyl group, an aryl group, a heterocyclic group, an ethenyl group,an ethynyl group, an amino group, an acyl group, a cyano group, analkoxy group, an aryloxy group, an alkylthio group, or an arylthiogroup, each of which may have a substituent, and any one of R³¹, . . . ,or R³⁵ is a hydrogen atom. R⁵¹ and R⁵² each independently represent analkyl group, an aryl group, a heteroaryl group, an amino group, an acylgroup, an alkoxy group, an aryloxy group, an alkylthio group, or anarylthio group, each of which may have a substituent. Q¹ and Q² eachindependently represent an aromatic hydrocarbon ring or an aromaticheterocyclic ring, each of which may have a substituent.

L¹ and L² each independently represent any one of Formulae (L-1) to(L-4).

In the formulae, R¹¹¹ to R¹¹⁶ each independently represent a hydrogenatom, a halogen atom, an alkyl group, an aryl group, a heterocyclicgroup, an ethenyl group, an ethynyl group, an amino group, an acylgroup, an alkoxy group, an aryloxy group, an alkylthio group, or anarylthio group, each of which may have a substituent. A represents —O—,—S—, or —NH—.

R¹¹ and R¹⁵ are each independently preferably an alkyl group, an arylgroup, a heterocyclic group, an ethenyl group, an ethynyl group, or anamino group, more preferably that as defined in R⁴¹ and R⁴², that is, anaryl group, a heterocyclic group, an ethenyl group, or an ethynyl group,and still more preferably an aryl group, an ethenyl group, or an ethynylgroup. From the viewpoint of improving a quantum yield, an aryl group ismore preferable, and from the viewpoint of increasing a wavelength, anethenyl group or an ethynyl group is more preferable. In the case ofbeing an aryl group, it is preferred to have at least one substituent atthe ortho or meta position of the aryl group, and it is more preferredto have at least one substituent at the ortho position of the arylgroup. The number of the substituent substituting for the aryl group ispreferably 1 to 3 and more preferably 2 or 3. The substituentsubstituting for the aryl group is preferably an alkyl group, morepreferably a methyl group, an isopropyl group, or a t-butyl group, andstill more preferably a methyl group.

<Specific Examples of Compounds Represented by Formulae (1) to (6)>

Specific examples of the compounds represented by Formulae (1) to (6)are shown below. Me represents a methyl group, Et represents an ethylgroup, and iPr represents an isopropyl group.

A labeled particle may be a labeled particle containing at least onekind of energy donor compound, at least one kind of energy acceptorcompound, and a particle, and in such a case, at least one kind of theenergy donor compound or the energy acceptor compound may be thecompound represented by Formula (1).

In another example of the present invention, a luminescent labeledparticle contains the compound represented by Formula (1) as one of anenergy donor compound or an energy acceptor compound, and a compoundrepresented by Formula (10) as the other of an energy donor compound oran energy acceptor compound. That is, the luminescent labeled particlemay be a luminescent labeled particle containing the compoundrepresented by Formula (1) as the energy donor compound and the compoundrepresented by Formula (10) as the energy acceptor compound, or may be aluminescent labeled particle containing the compound represented byFormula (1) as the energy acceptor compound and the compound representedby Formula (10) as the energy donor compound.

<Compound Represented by Formula (10)>

In Formula (10), m1 and m2 each independently represent an integer of 0to 4, and any one of m1 or m2 is at least one. M represents a metalloidatom or a metal atom. R¹, R², and R³ each independently represent ahydrogen atom, an alkyl group, an aryl group, a heterocyclic group, anethenyl group, an ethynyl group, an acyl group, an alkoxy group, anaryloxy group, an alkylthio group, or an arylthio group, each of whichmay have a substituent. Y¹ and Y² each independently represent a halogenatom, an alkyl group, an aryl group, a heterocyclic group, a hydroxygroup, an alkoxy group, an aryloxy group, an alkylthio group, anarylthio group, an ethenyl group, or an ethynyl group, each of which mayhave a substituent, and Y¹ and Y² may be linked to each other to form aring. Ar¹¹ and Ar¹² each independently represent an aromatic ring whichmay have a substituent. Z¹ and Z² each independently represent an arylgroup, a heterocyclic group, or an amino group, each of which may have asubstituent. In the case where m1 is two or more, a plurality of Z¹'smay be the same group or different groups, and in the case where m2 istwo or more, a plurality of Z²'s may be the same group or differentgroups.

In Formula (10), m1 and m2 each independently represent an integer of 0to 4, and preferably both m1 and m2 are one or more. m1 and m2 may bethe same integer or different integers, and are preferably the sameinteger. Preferably, m1 and m2 are each independently one or two, morepreferably, both m1 and m2 are one or two, and particularly preferablyboth m1 and m2 are one.

In Formula (10), M represents a metalloid atom or a metal atom,preferably a metalloid atom, and particularly preferably a boron atom.

In Formula (10), R¹, R², and R³ each independently represent a hydrogenatom, an alkyl group, an aryl group, a heterocyclic group, an ethenylgroup, an ethynyl group, an acyl group, an alkoxy group, an aryloxygroup, an alkylthio group, or an arylthio group, each of which may havea substituent.

Preferably, R¹ and R² each independently represent an aryl group or aheterocyclic group, each of which may have a substituent.

R¹ and R² may be the same as or different from each other, and arepreferably the same as each other.

R¹ and R² are not linked to each other to form a ring.

Preferably, R³ represents a hydrogen atom, an alkyl group, an arylgroup, or a heterocyclic group, each of which may have a substituent.More preferably, R³ is a hydrogen atom.

In Formula (10), Y¹ and Y² each independently represent a halogen atom,an alkyl group, an aryl group, a heterocyclic group, a hydroxy group, analkoxy group, an aryloxy group, an alkylthio group, an arylthio group,an ethenyl group, or an ethynyl group, each of which may have asubstituent, and Y¹ and Y² may be linked to each other to form a ring.

Preferably, Y¹ and Y² each independently represent a halogen atom, analkyl group, an aryl group, a hydroxy group, an alkoxy group, or anaryloxy group, each of which may have a substituent, and Y¹ and Y² maybe linked to each other to form a ring.

More preferably, Y¹ and Y² are each independently halogen atoms.

Still more preferably, Y¹ and Y² are fluorine atoms.

Y¹ and Y² may be the same as or different from each other, and arepreferably the same as each other.

In Formula (10), Ar¹ and Ar² each independently represent an aromaticring which may have a substituent.

Preferably, Ar¹ and Ar² each represent a benzene ring.

In Formula (10), Z¹ to Z² each independently represent a halogen atom,an alkyl group, an aryl group, a heterocyclic group, an ethenyl group,an ethynyl group, an acyl group, an alkoxy group, an aryloxy group, analkylthio group, an arylthio group, or an amino group, each of which mayhave a substituent. In the case where m1 is two or more, a plurality ofZ¹'s may be the same group or different groups, and in the case where m2is two or more, a plurality of Z²'s may be the same group or differentgroups.

Preferably, Z¹ and Z² each independently represent an aryl group whichmay have a substituent.

More preferably, Z¹ and Z² each independently represent a phenyl group,a naphthyl group, or an anthryl group, each of which may have asubstituent.

Preferably, in the case where m1 is two or more, a plurality of Z¹'s arethe same group.

Preferably, in the case where m2 is two or more, a plurality of Z²'s arethe same group.

It is preferred that the compound represented by Formula (2) does nothave acidic groups, such as a carboxylic acid group, a phosphoric acidgroup, and a sulfonic acid group, in a molecule.

<As to Compound Represented by Formula (10A)>

A preferred example of the compound represented by Formula (10) is acompound represented by Formula (10A).

In Formula (10A), Y¹ to Y² each independently represent a halogen atom,an alkyl group, an aryl group, a heterocyclic group, a hydroxy group, analkoxy group, an aryloxy group, an alkylthio group, or an arylthiogroup, an ethenyl group, or an ethynyl group, each of which may have asubstituent. Examples of the substituent include the substituentsdescribed in Substituent group A.

Preferably, Y¹ and Y² each independently represent halogen atoms.

Particularly preferably, Y¹ and Y² are fluorine atoms.

In Formula (10A), R³ represents a hydrogen atom, an alkyl group, an arylgroup, a heterocyclic group, an ethenyl group, an ethynyl group, or anacyl group, each of which may have a substituent.

Preferably, R³ represents a hydrogen atom, an alkyl group, an arylgroup, or a heterocyclic group, each of which may have a substituent.

More preferably, R³ is a hydrogen atom.

In Formula (10A), Ar³ and Ar⁴ each independently represent an aryl groupor a heterocyclic group, each of which may have a substituent. Examplesof the substituent include the substituents described in Substituentgroup A.

In Formula (10A), R³⁴ to R⁴¹ each independently represent a hydrogenatom, a halogen atom, an alkyl group, an aryl group, a heterocyclicgroup, an ethenyl group, an ethynyl group, an acyl group, an alkoxygroup, an aryloxy group, an alkylthio group, an arylthio group, or anamino group, each of which may have a substituent. Examples of thesubstituent include the substituents described in Substituent group A.

In Formula (10A), at least one of R³⁴, . . . , or R⁴¹ is preferably anaryl group which may have a substituent.

More preferably, at least one of R³⁴, . . . , or R³⁷ is an aryl groupwhich may have a substituent, and at least one of R³⁸, . . . , or R⁴¹ isan aryl group which may have a substituent.

More preferably, at least one of R³⁴, . . . , or R⁴¹ is a grouprepresented by Formula (11). Still more preferably, at least one of R³⁴,. . . , or R³⁷ is a group represented by Formula (11), and at least oneof R³⁸, . . . , or R⁴¹ is a group represented by Formula (11).

In Formula (11), R²⁰¹ to R²⁰⁵ are each a hydrogen atom, a halogen atom,an alkyl group, an aryl group, a heterocyclic group, an ethenyl group,an ethynyl group, an acyl group, an alkoxy group, an aryloxy group, analkylthio group, an arylthio group, or an amino group, and at least oneof R²⁰¹, . . . , or R²⁰⁵, is an atom or group other than a hydrogenatom. R²⁰¹ and R²⁰² may be linked to each other to form a ring, R²⁰² andR²⁰³ may be linked to each other to form a ring, R²⁰³ and R²⁰⁴ may belinked to each other to form a ring, and R²⁰⁴ and R²⁰⁵ may be linked toeach other to form a ring.

According to another preferred aspect, at least one of R³⁴, . . . , orR⁴¹ is a group represented by Formula (12). More preferably, at leastone of R³⁴, . . . , or R³⁷ is a group represented by Formula (12), andat least one of R³⁸, . . . , or R⁴¹ is a group represented by Formula(12).

In Formula (12), R¹⁰¹ represents a hydrogen atom, an alkyl group, anaryl group, a heterocyclic group, an ethenyl group, an ethynyl group, oran acyl group, each of which may have a substituent. Examples of thesubstituent include the substituents described in Substituent group A.Ar¹⁰¹ represents an aryl group or a heterocyclic group, each of whichmay have a substituent. Examples of the substituent include thesubstituents described in Substituent group A. Ar¹⁰¹ and R¹⁰¹ may belinked to each other to form a ring.

It is preferred that the compound represented by Formula (10) does nothave acidic groups, such as a carboxylic acid group, a phosphoric acidgroup, and a sulfonic acid group, in a molecule.

<Specific Examples of Compound Represented by Formula (10) or Formula(10A)>

Specific examples of the compound represented by Formula (10) or Formula(10A) are shown below. Me represents a methyl group, Bu represents ann-butyl group, and Ph represents a phenyl group.

<As to Specific Examples of Combination of Energy Donor Compound andEnergy Acceptor Compound>

Specific examples of a combination of an energy donor compound and anenergy acceptor compound are shown below.

TABLE 1 Donor Acceptor Donor Acceptor Donor Acceptor F-2 F-43 F-16 F-64F-37 F-43 F-2 F-44 F-16 F-69 F-37 F-44 F-2 F-46 F-16 F-70 F-37 F-46 F-2F-51 F-16 F-71 F-37 F-51 F-2 F-55 F-16 F-72 F-37 F-55 F-2 F-69 F-24 F-43F-65 F-43 F-16 F-38 F-24 F-44 F-65 F-44 F-16 F-39 F-24 F-46 F-65 F-46F-16 F-41 F-24 F-51 F-65 F-51 F-16 F-42 F-24 F-55 F-65 F-55 F-16 F-43F-24 F-69 F-65 F-69 F-16 F-45 F-27 F-43 F-66 F-43 F-16 F-46 F-27 F-44F-66 F-44 F-16 F-48 F-27 F-46 F-66 F-46 F-16 F-49 F-27 F-51 F-66 F-51F-16 F-50 F-27 F-55 F-66 F-55 F-16 F-51 F-27 F-69 F-66 F-69 F-16 F-52F-29 F-43 F-67 F-43 F-16 F-53 F-29 F-44 F-67 F-44 F-16 F-54 F-29 F-46F-67 F-46 F-16 F-55 F-29 F-51 F-67 F-51 F-16 F-56 F-29 F-55 F-67 F-55F-16 F-57 F-29 F-69 F-67 F-69 F-16 F-58 F-33 F-43 F-68 F-43 F-16 F-59F-33 F-44 F-68 F-44 F-16 F-60 F-33 F-46 F-68 F-46 F-16 F-61 F-33 F-51F-68 F-51 F-16 F-62 F-33 F-55 F-68 F-55 F-16 F-63 F-33 F-69 F-68 F-69

TABLE 2 Donor Acceptor Donor Acceptor Donor Acceptor E-4 F-43 E-16 F-64F-16 E-24 E-4 F-44 E-16 F-69 F-19 E-24 E-4 F-46 E-16 F-70 F-20 E-24 E-4F-51 E-16 F-71 F-21 E-24 E-4 F-55 E-16 F-72 F-23 E-24 E-4 F-69 E-20 F-43F-24 E-24 E-16 F-38 E-20 F-44 F-25 E-24 E-16 F-39 E-20 F-46 F-26 E-24E-16 F-41 E-20 F-51 F-27 E-24 E-16 F-42 E-20 F-55 F-28 E-24 E-16 F-43E-20 F-69 F-29 E-24 E-16 F-45 E-57 F-43 F-30 E-24 E-16 F-46 E-57 F-44F-31 E-24 E-16 F-48 E-57 F-46 F-32 E-24 E-16 F-49 E-57 F-51 F-33 E-24E-16 F-50 E-57 F-55 F-34 E-24 E-16 F-51 E-57 F-69 F-36 E-24 E-16 F-52F-1 E-24 F-37 E-24 E-16 F-53 F-2 E-24 F-65 E-24 E-16 F-54 F-3 E-24 F-66E-24 E-16 F-55 F-4 E-24 F-67 E-24 E-16 F-56 F-5 E-24 F-68 E-24 E-16 F-57F-6 E-24 F-2 E-17 E-16 F-58 F-7 E-24 F-16 E-17 E-16 F-59 F-8 E-24 F-33E-17 E-16 F-60 F-12 E-24 F-65 E-17 E-16 F-61 F-13 E-24 F-66 E-17 E-16F-62 F-14 E-24 F-67 E-17 E-16 F-63 F-15 E-24 F-68 E-17

Regarding the selection of an energy donor compound and an energyacceptor compound, a compound with absorption in a short wavelength isthe energy donor compound, a compound with absorption in a longwavelength is the energy acceptor compound, and in the case where theemission of the energy donor compound and the absorption of the energyacceptor compound overlap each other even a little, the compounds may beusable in the luminescent labeled particle. It is preferred that anabsorption maximum wavelength of the energy acceptor compound is on thelonger wavelength side by about 10 to 100 nm than an absorptionwavelength of the energy donor compound. It is more preferred that anabsorption maximum wavelength of the energy acceptor compound is on thelonger wavelength side by about 10 to 70 nm than an absorptionwavelength of the energy donor compound.

How longer the emission wavelength of the energy donor compound is thanabsorption wavelength (the size of the Stokes shift) varies depending oncompounds, and thus it is difficult to be defined uniformly. However,since the compound represented by Formula (1) has maximum emission in awavelength which is longer than the absorption maximum wavelength byabout 30 nm, and has an emission spectrum in a range of the wavelengthto a wavelength longer than the wavelength by about 100 nm, it isassumed that an energy transfer system can be realized by combined useof an acceptor compound with absorption in the vicinity of the emissionspectrum.

The absorption wavelength of each compound not only can be measuredafter synthesizing the compounds, but also can be predicted fromcalculation by Gaussian or the like. Additionally, it is possible toestimate a combination of the energy donor compound and the energyacceptor compound from the relationship between the calculated values.

In the present invention, the size of the Stokes shift is preferably 25nm or more, more preferably 30 nm or more, still more preferably 35 nmor more, even more preferably 40 nm or more, even still more preferably45 nm or more, particularly preferably 50 nm or more, and mostpreferably 60 nm or more. An upper limit of the size of the Stokes shiftis not particularly limited, but is generally 150 nm or less.

<Amount of Use of Compounds Represented by Formulae (1) to (6)>

There is no particular limitation on the content of the compoundrepresented by Formula (1) for the particles used in the presentinvention (that is, the particles before addition of the compoundrepresented by Formula (1)) as long as the effect of the presentinvention is not impaired, but the content is preferably 0.5 μmol/g to400 μmol/g, more preferably 1 μmol/g to 300 μmol/g, still morepreferably 2 μmol/g to 200 μmol/g, and particularly preferably 3 μmol/gto 100 μmol/g.

There is no particular limitation on the content of the compoundsrepresented by Formulae (1) to (6) for the particles used in the presentinvention (that is, the particles before addition of the compoundsrepresented by Formulae (1) to (6)) as long as the effect of the presentinvention is not impaired, but the content is preferably 0.1% by mass to30% by mass, more preferably 0.2% by mass to 20% by mass, still morepreferably 0.3% by mass to 10% by mass, and particularly preferably 0.4%by mass to 8% by mass.

In the luminescent labeled particle, at least one kind of compoundrepresented by Formulae (1) to (6) is used, but two or more kinds ofcompounds represented by Formulae (1) to (6) may be used. In the casewhere two or more kinds of compounds represented by Formulae (1) to (6)are used, it is preferred that the total amount of the compounds fallswithin the above range.

In the case of using the combination of the energy donor compound andthe energy acceptor compound, the molar ratio of the energy donorcompound to the energy acceptor compound is preferably 1:10 to 20:1,more preferably 1:10 to 10:1, and still more preferably 1:5 to 10:1.

In the case where at least one kind of compound represented by Formula(1) is used as the energy donor compound and at least one kind ofcompound represented by Formula (1) is used as the energy acceptorcompound, two or more kinds of compounds represented by Formula (1) maybe used as the energy donor compound, and two or more kinds of compoundsrepresented by Formula (1) may be used as the energy acceptor compound.In the above case, it is preferred that the total amount of thecompounds represented by Formula (1) to be used falls within the aboverange.

<Method for Producing Compounds Represented by Formulae (1) to (6)>

The compounds represented by Formulae (1) to (6) can be produced, forexample, according to a synthesis scheme shown in Examples which will bedescribed later.

As an example, the synthesis of Compound (1) is outlined below.3-Ethyl-2,4-dimethylpyrrole and trifluoroacetic acid are added to amixture of 3,5-bis(trifluoromethyl)benzaldehyde and dichloromethanewhile cooling with water, followed by stirring at room temperature,chloranil is added while cooling with water, followed by stirring atroom temperature, and diisopropylethylamine is added dropwise whilecooling with water, followed by stirring at room temperature.Subsequently, a boron trifluoride-diethyl ether complex is addeddropwise while cooling with water, and the reaction is carried out bystirring the mixture at room temperature, whereby Compound (1-A) can besynthesized. Subsequently, Compound (1-A), 115 mg of2,4,6-trimethylbenzaldehyde, and dehydrated toluene are mixed andstirred at room temperature. Piperidine and one piece ofp-toluenesulfonic acid monohydrate are added, and the mixture is stirredwhile distilling off the solvent. After allowing to cool, dehydratedtoluene is added and the reaction is carried out by stirring the mixturewhile distilling off the solvent, whereby Compound (1) can be produced.

As another example, Compound (3) can be produced through Compound (3-A),Compound (3-B), and Compound (3-C) from3,5-bis(trifluoromethyl)benzaldehyde and 2,4-dimethylpyrrole as startingcompounds according to the synthesis scheme of <Synthesis Example 2> inExamples which will be described later.

Compound (1) and Compound (3) are within the definition of the compoundrepresented by Formula (1). The compound represented by Formula (1)other than Compound (1) and Compound (3) can also be produced bysubstituting the compound used in the reaction with a compound having asubstituent corresponding to a desired target compound represented byFormula (1).

<Method for Producing Compound Represented by Formula (10)>

The compound represented by Formula (10) can be produced, for example,according to the following synthesis scheme.

The definitions of R¹ and Z¹ in the above synthesis scheme are the sameas the definitions of R¹ and Z¹ in formula (10).

Compound A-30 can be synthesized by reacting Compound A-10 with CompoundA-20 according to the method described in Macromolecules 2010, 43, 193to 200. Then, Compound A-30, a compound represented by a formula ofZ¹—B(OH)₂, and cesium fluoride (CsF) are added to a mixed solution ofdimethoxyethane (DME) and water, and vacuum drawing and nitrogensubstitution are repeated for degassing. Compound D-10 can be producedby adding palladium acetate (Pd(OAc)₂) and2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl (SPhos) thereto, raisingthe temperature, and performing the reaction under reflux for apredetermined time (for example, 2 to 24 hours).

Compound D-10 is within the definition of the compound represented byFormula (10). The compound represented by Formula (10) other thanCompound D-10 can also be produced by substituting any one or more ofCompound A-10, Compound A-20, or the compound represented by a formulaof Z¹—B(OH)₂ with corresponding compounds.

<Particle>

Labeled particles include particles. The material and form of theparticles are not particularly limited, and for example, organic polymerparticles such as polystyrene beads or inorganic particles such as glassbeads can be used. Specific examples of the material of the particlesinclude a homopolymer obtained by polymerizing a monomer such asstyrene, methacrylic acid, glycidyl (meth)acrylate, butadiene, vinylchloride, vinyl acetate acrylate, methyl methacrylate, ethylmethacrylate, phenyl methacrylate, or butyl methacrylate, and acopolymer obtained by polymerizing two or more monomers. A latex inwhich the homopolymer or the copolymer is uniformly suspended may alsobe used. Examples of the particles include other organic polymerpowders, inorganic substance powders, microorganisms, blood cells, cellmembrane fragments, liposomes, and microcapsules. Latex particles arepreferable as particles.

In the case where latex particles are used, specific examples of thematerial of the latex include polystyrene, a styrene-acrylic acidcopolymer, a styrene-methacrylic acid copolymer, a styrene-glycidyl(meth)acrylate copolymer, a styrene-styrene sulfonate copolymer, amethacrylic acid polymer, an acrylic acid polymer, anacrylonitrile-butadiene-styrene copolymer, a vinyl chloride-acrylic acidester copolymer, and polyvinyl acetate acrylate. As the latex, acopolymer containing at least styrene as a monomer is preferable, and acopolymer of styrene and acrylic acid or methacrylic acid isparticularly preferable. The method for preparing the latex is notparticularly limited, and the latex can be prepared by anypolymerization method. However, in the case where the luminescentlabeled particle is used by labeling with an antibody, the presence of asurfactant makes it difficult to immobilize the antibody. Therefore, forthe preparation of a latex, emulsifier-free emulsion polymerization,that is, emulsion polymerization without using an emulsifier such as asurfactant is preferable.

<Luminescent Labeled Particle>

By including the compound represented by Formula (1), the luminescentlabeled particle in the present invention has an emission maximumwavelength in the long wavelength range of 680 nm or longer and exhibitsa high quantum yield.

The emission maximum wavelength refers to a wavelength at which theabsorbance becomes the largest in the absorption spectrum.

The emission maximum wavelength of the luminescent labeled particle ispreferably 680 nm or longer, more preferably 700 nm or longer, andparticularly preferably 720 nm or longer. An upper limit of the emissionmaximum wavelength of the luminescent labeled particle of the presentinvention is not particularly limited, but is preferably 900 nm orshorter and more preferably 800 nm or shorter.

The emission maximum wavelength of the luminescent labeled particle canbe measured using a commercially available fluorescencespectrophotometer, and for example, can be measured using a fluorescencespectrophotometer RF-5300PC manufactured by Shimadzu Corporation.

The quantum yield of the luminescent labeled particles is the ratio ofthe number of photons emitted as fluorescence to the number of photonsabsorbed by luminescent labeled particles.

The quantum yield of the luminescent labeled particles is preferably0.25 or more, more preferably 0.4 or more, still more preferably 0.5 ormore, even more preferably 0.6 or more, and particularly preferably 0.7or more. An upper limit of the quantum yield is not particularlylimited, but generally is 1.0 or less.

The quantum yield of the luminescent labeled particles can be measuredusing a commercially available quantum yield measuring apparatus, andfor example, can be measured using an absolute PL quantum yieldspectrometer C9920-02 manufactured by Hamamatsu Photonics K.K.

(Method for Measuring Average Particle Diameter (Average Particle Size)of Luminescent Labeled Particles)

The average particle diameter of the luminescent labeled particlesvaries depending on the material of the particles, the concentrationrange for measuring the test substance, the measuring device, and thelike, but is preferably in the range of 0.001 to 10 μm (more preferably0.01 to 1 μm), more preferably in the range of 30 to 500 nm, still morepreferably in the range of 50 to 300 nm, particularly preferably in therange of 80 to 200 nm, and most preferably in the range of 100 to 150nm. The average particle diameter of the luminescent labeled particlesthat can be used in the present invention can be measured with acommercially available particle size distribution meter or the like. Asa method for measuring the particle size distribution, opticalmicroscopy, confocal laser microscopy, electron microscopy, atomic forcemicroscopy, static light scattering method, laser diffraction method,dynamic light scattering method, centrifugal sedimentation method,electric pulse measurement method, chromatography method, ultrasonicattenuation method, and the like are known, and apparatusescorresponding to the respective principles are commercially available.Among these measurement methods, it is preferred to measure the averageparticle diameter of the luminescent labeled particles using a dynamiclight scattering method from the viewpoint of the particle size rangeand ease of measurement. Examples of commercially available measuringapparatuses using dynamic light scattering include NANOTRAC UPA (NikkisoCo., Ltd.), dynamic light-scattering particle size analyzer LB-550(HORIBA, Ltd.), fiber-optics particle analyzer FPAR-1000 (OtsukaElectronics Co., Ltd.), and the like. In the present invention, theaverage particle diameter is obtained as a median diameter (d=50)measured at 25° C. under the conditions of a viscosity of 0.8872 CP anda refractive index of water of 1.330.

<Method for Producing Luminescent Labeled Particles>

The method for producing the luminescent labeled particles is notparticularly limited, but the luminescent particles can be produced bymixing particles with at least one kind of compound represented byFormula (1). For example, the luminescent labeled particles can beprepared by adding the compound represented by Formula (1) to particlessuch as latex particles. More specifically, the luminescent labeledparticles can be produced by adding a solution containing the compoundrepresented by Formula (1) to a dispersion liquid of particlescontaining at least one of water or a water-soluble organic solvent(tetrahydrofuran, methanol, or the like) and stirring the mixture.

In the present invention, a dispersion liquid containing theabove-described luminescent labeled particle of the present inventionmay be prepared.

The dispersion liquid can be produced by dispersing the luminescentlabeled particles of the present invention in a dispersion medium.Examples of the dispersion medium include water, an organic solvent, anda mixture of water and an organic solvent. An alcohol such as methanol,ethanol, or isopropanol, an ether-based solvent such as tetrahydrofuran,or the like can be used as the organic solvent.

The concentration of the solid content of the luminescent labeledparticles in the dispersion liquid is not particularly limited, but isgenerally 0.1% to 20% by mass, preferably 0.5% to 10% by mass, and morepreferably 1% to 5% by mass.

(Modification of Luminescent Labeled Particle by First BindingSubstance)

The method for immobilizing the first binding substance on theluminescent labeled particle is described, for example, inJP2000-206115A or the protocol attached to FluoSpheres (registeredtrademark) polystyrene microsphere F8813 of Thermo Fisher ScientificInc., and any known method for preparing a reagent for animmunoagglutination reaction can be used. In addition, as a principle ofimmobilizing an antibody as a binding substance on particles, anyprinciple of physical adsorption or a chemical bond by a covalent bondcan be adopted. As a blocking agent (that is, the first blocking agent)covering a particle surface which is not coated with the antibody afterimmobilizing the antibody on the particle, for example, albumin (such asBSA), skim milk, casein, a soybean-derived component, a fish-derivedcomponent, polyethylene glycol, or the like, and commercially availableblocking agents for an immune reaction or the like containing thesubstances as well as substances having the same property as that of thesubstances can be used. These blocking agents can be subjected topretreatment such as partial modification with heat, acid, alkali, orthe like, as necessary. Furthermore, as the first blocking agent, anantibody (globulin) which is not capable of binding to the measurementtarget substance or a protein (Protein A and Protein G) which is notused in a test area can be used.

A specific method for immobilizing an antibody on particles isexemplified below. An antibody solution of which concentration isadjusted to 0.01 to 20 mg/mL is added to a liquid in which the particlesare dispersed such that the concentration of the solid content of theparticles becomes 0.1% to 10% by mass, and mixing is performed. Stirringis continued for 5 minutes to 48 hours under a condition of atemperature of 4° C. to 50° C. Next, the particle and the solution areseparated by centrifugation or other methods to sufficiently removeantibodies not bound to the particle contained in the solution. Then, anoperation of washing the particle with a buffer solution is repeated 0to 10 times. It is preferred that after carrying out an operation ofmixing the particle and the antibody and binding the antibody to theparticle, a portion of the particle surface to which the antibody is notbound is protected using a blocking agent such as the components whichdo not participate in the antigen-antibody reaction, preferably protein,and more preferably globulin, albumin, BLOCKACE (registered trademark),skim milk, and casein.

In the case where the antigen, the antibody, or the like is immobilizedon the particle, a stabilizer can be added, as necessary. The stabilizeris not particularly limited as long as the stabilizer stabilizes anantigen or an antibody, like a synthetic polymer or a natural polymer,such as polysaccharides or sucrose, and commercially availablestabilizers such as Immunoassay Stabilizer (Advanced BiotechnologiesInc.) can also be used.

The labeled particle having the first binding substance is contained inthe kit according to the embodiment of the present invention, and anaspect in which the labeled particle is contained in a container, forexample, a cup, which is a part of the kit is preferable. In this case,the measurement target substance in the biological sample can be boundto the first binding substance by injecting the biological sample into acontainer containing the labeled particle, and mixing and stirringcomponents.

(Substrate)

In the present invention, in order to achieve high-sensitivemeasurement, it is preferred to adopt a measurement method forperforming surface plasmon fluorescence (SPF) detection described later.As a substrate in this case, it is preferred to use a substrate having ametal film on a surface. A metal constituting the metal film is notparticularly limited as long as the metal can cause surface plasmonresonance. Preferably, free-electron metals such as gold, silver,copper, aluminum, or platinum can be mentioned, and gold is particularlypreferable. In the case where gold is used, the detection area describedlater is on the gold film. The metals can be used alone or in acombination thereof. Further, in consideration of the adhesiveness tothe substrate, an intervening layer including chromium or the like maybe provided between the substrate and the layer including metal.Thickness of the metal film is randomly determined, but for example, ispreferably 1 nm or more and 500 nm or less, and particularly preferably10 nm or more and 200 nm or less. In the case where the thicknessexceeds 500 nm, a surface plasmon phenomenon of a medium cannot bedetected sufficiently. Moreover, in the case of providing an interveninglayer which includes chromium or the like, it is preferred thatthickness of the intervening layer is 0.1 nm or more and 10 nm or less.

The formation of the metal film may be carried out by a conventionalmethod, and can be carried out, for example, by a sputtering method, avapor deposition method, an ion plating method, an electroplatingmethod, a non-electrolytic plating method, or the like. In order toprovide a mixed layer of a substrate material and a metal film andimprove the adhesiveness of the metal film, it is preferred to preparethe metal film by the sputtering method. In this case, thickness of themixed layer of the substrate material and the metal film is notparticularly limited as long as sufficient adhesiveness can be ensured,and 10 nm or less is preferable.

The metal film is preferably disposed on the substrate. Herein,“disposed on the substrate” includes a case where the metal film isdisposed to be in direct contact with the substrate, and a case wherethe metal film is disposed not in direct contact with the substrate butin contact with the substrate through other layers. The material of thesubstrate that can be used in the present invention is, for example,optical glass such as BK7 (borosilicate glass), which is a type ofgeneral optical glass, or synthetic resin, specifically a substanceformed of a material transparent to laser light, such as polymethylmethacrylate, polyethylene terephthalate, polycarbonate, or acycloolefin polymer can be used. Such a substrate is preferably amaterial that does not exhibit anisotropy with respect to polarizationand has excellent processability.

As a preferred aspect of the substrate for SPF detection, a substrate inwhich a gold film is vapor-deposited on polymethyl methacrylate (PMMA)can be mentioned.

The substrate comprises a detection area having a second bindingsubstance that is capable of binding to any one of the measurementtarget substance or the first binding substance.

(Second Binding Substance)

A second binding substance is a substance capable of binding to themeasurement target substance or a substance capable of binding to thefirst binding substance. In the case where quantification is performedby a sandwich assay method, a substance capable of binding to themeasurement target substance can be used as the second bindingsubstance. In the case where quantification is performed by acompetition method, a substance capable of binding to the first bindingsubstance can be used as the second binding substance. In the presentinvention, quantification is preferably performed by a competitionmethod, and it is preferred to use a substance capable of binding to thefirst binding substance as the second binding substance.

The second binding substance is not particularly limited, but preferredexamples thereof include an antigen, an antibody, or a complex thereof,an antigen is preferable, and particularly preferably a measurementtarget substance (this is a substance capable of binding to the firstbinding substance) is used as the second binding substance.

In the case where the measurement target substance is used as the secondbinding substance, the second binding substance is preferably aconjugate of the measurement target substance and a carrier. The carriermeans a substance to which a plurality of molecules of the measurementtarget substance can be bound. As an example of a preferred carrier,proteins or the like are mentioned, and among them, specifically, bovineserum albumin or the like can be mentioned.

In the case where the measurement target substance is bile acid, thesecond binding substance particularly preferably includes cholic acidand/or a cholic acid-albumin conjugate, deoxycholic acid and/or adeoxycholic acid-albumin conjugate, and chenodeoxycholic acid and/or achenodeoxycholic acid-albumin conjugate. In the case where themeasurement target substance is progesterone, the second bindingsubstance is preferably a progesterone-albumin conjugate.

(Method for Immobilizing Second Binding Substance on Substrate)

A method for immobilizing a second binding substance on a substrate isdescribed in, for example, Tech Notes Vols. 2 to 12 provided by NuncCorporation and all known methods for preparing a general Enzyme-linkedimmunosorbent assay (ELISA) reagent can be used. In addition, surfacemodification may be performed by placing a self-assembled monolayer(SAM) or the like on a substrate, and as a method for immobilizing thesecond binding substance on the substrate, any method using physicaladsorption or a chemical bond by a covalent bond can be adopted. As ablocking agent (second blocking agent) covering the substance surfacewhich is not coated with the second binding substance after immobilizingthe second binding substance on the substrate, known substances, forexample, BSA, globulin, skim milk, casein, a soybean-derived component,a fish-derived component, polyethylene glycol, or the like, andcommercially available blocking agents for an immune reaction or thelike containing the substances as well as substances having the sameproperty as that of the substances can be used. These blocking agentscan be subjected to pretreatment such as partial modification with heat,acid, alkali, or the like, as necessary.

(Detection Area <Test Area>)

In the present invention, a test area can be provided on the substrateto detect the presence or absence of the measurement target substance inthe biological sample. In this test area, for example, an antigen can bequantified by capturing an antigen which is a measurement targetsubstance and detecting and quantifying the amount of labels bound tothe antigen. Alternatively, the antigen can be quantified by a method inwhich only the labels bound to the antigen is caused not to be bound,only labels not bound to the antigen is captured, and the amount oflabels bound to the antigen is calculated. This detection method isreferred to as a competition method and herein, the substrate relatingto the competition method will be described.

It is preferred that the test area of the substrate has a site forreacting with the binding substance (for example, antibody) present onthe labeled particle. As a preferred aspect of the present invention, anaspect in which the antigen present in the biological sample is on thetest area of the substrate is preferable. In this case, the antigen andBSA are reacted in the presence of a condensing agent to prepare anantigen-BSA conjugate, and a test area can be prepared by adsorbing theconjugate onto the test area. The antigen-BSA conjugate which is themeasurement target substance can be bound to the test area on asubstrate by a method in which the conjugate is dissolved in a buffersolution, and the resultant is spotted on the substrate and allowed tostand for a predetermined time, the supernatant is sucked, and drying isperformed.

(Reference Area <Control Area>)

In the present invention, in order to minimize influence of themeasurement environment, particularly the measurement temperature, asmuch as possible, a control area is provided on the substrate, and theinformation on the test area is standardized by the information on thecontrol area, thereby enabling the environmental dependency to besuppressed extremely low. The control area is preferably designed to becapable of binding to all the labels regardless of the amount of themeasurement target substance present in the biological sample to beused. It is preferred to provide an antibody that interacts with all theantibodies present on the labeled particle. By designing in this mannerto standardize the information on the test area by the information onthe control area, for example, even in the case where the flow of thebiological sample or the reaction rate is affected in the lowtemperature environment, such influence can be cancelled by thestandardization, and thus it becomes possible to obtain a result that isalways precise and not affected by the measurement environment.

An antibody to be present in the control area preferably has a functionof recognizing a binding substance (for example, antibody) present onthe labeled particle, in the case where the antibody is derived from amouse, an anti-mouse antibody is preferable, and in the case where theantibody on the labeled particle is derived from a goat, an anti-goatantibody is preferable. These antibodies on the control area can bebound to a substrate by a method in which the antibodies are dissolvedin a buffer solution, and the resultant is spotted on the substrate andallowed to stand for a predetermined time, the supernatant is sucked,and drying is performed.

(Blocking Agent)

For example, in a competition method, not only a negative biologicalsample which does not contain a measurement target substance but also abiological sample which becomes negative by reacting to even a positivebiological sample which contains a measurement target substance arepresent, and the solution to the problem of deviation at a high value isrecognized as an issue. The cause of the false negative is not clear,but it is considered that the presence of labeled particles which areoriginally not desired to bind due to nonspecific interaction betweenthe labeled particle surface not covered with the antibody and thedetection area (test area) is one of the causes. Moreover, in the casewhere the same substance as the substance present on the test area ispresent on the surface of the labeled particle, and a free antibody orthe like is present in the biological sample, even in the measurement ofa positive biological sample containing the measurement targetsubstance, the antibody may be detected as negative by binding to boththe substances present on the test area and the substance on the surfaceof the labeled particle.

In general, blocking with BSA is used to suppress nonspecific adsorptiononto a solid phase surface (for example, a labeled particle surface, anda gold film surface of a substrate).

As an immunoglobulin other than the immunoglobulin capable of binding tothe measurement target substance, specifically, an antiserum preparedfrom a serum of an animal immunized with an antigen different from themeasurement target substance, an immunoglobulin fraction purified fromthe antiserum, a monoclonal antibody obtained by cell fusion usingspleen cells of an animal immunized with the measurement targetsubstance, or a fragment thereof [for example, F(ab′)₂, Fab, Fab′, orFv] can be used. Preparation of these antibodies can be performed by aconventional method. Furthermore, the antibody may be modified as in thecase of a chimeric antibody or the like, or a commercially availableantibody or an antibody prepared from an animal serum or culturesupernatant by known methods can be used.

(Antibody)

In the present invention, antibodies can be used regardless of animalspecies or subclasses thereof. For example, an antibody that can be usedin the present invention is an antibody derived from an organism inwhich an immune reaction can occur, such as mice, rats, hamsters, goats,rabbits, sheep, cows, or chickens, specific examples thereof includemouse IgG, mouse IgM, rat IgG, rat IgM, hamster IgG, hamster IgM, rabbitIgG, rabbit IgM, goat IgG, goat IgM, sheep IgG, sheep IgM, bovine IgG,bovine IgM, chicken IgY, and the like, and either polyclonal ormonoclonal antibody can be used. A fragmented antibody is a moleculederived from a complete antibody, having at least one antigen bindingsite, and is specifically Fab, F(ab′)₂, or the like. These fragmentedantibodies are molecules obtained by an enzyme or chemical treatment orby using genetic engineering techniques.

(Other Elements of Kit)

The kit according to the embodiment of the present invention is used ina method for measuring a measurement target substance, in the case wherethe measurement target substance is bile acid, the kit is a kit for bileacid measurement and diagnosis, and in the case where the measurementtarget substance is progesterone, the kit is a kit for progesteronemeasurement and diagnosis. In the present invention, in the case ofperforming measurement of a measurement target substance, the kitincludes a substrate on which a second binding substance is immobilized,and a sensor chip including a member holding labeled particles such asfluorescent particles, but may include various instruments orapparatuses used in measurement of a measurement target substance, suchas a surface plasmon excitation apparatus and a fluorescence measurementdevice. Furthermore, a sample containing a known amount of themeasurement target substance, an instruction manual, or the like may beincluded as an element of the kit.

[Method for Measuring Measurement Target Substance in Biological Sample]

The method for measuring a measurement target substance in a biologicalsample according to the embodiment of the present invention is a methodincluding a reaction step of reacting a biological sample with a labeledparticle having a first binding substance capable of binding to ameasurement target substance, a capturing step of capturing the labeledparticle on a substrate having a second binding substance capable ofbinding to any one of the measurement target substance or the firstbinding substance by bringing a reaction product obtained in thereaction step into contact with the substrate, and a label informationacquisition step of acquiring label information related to themeasurement target substance, in which the labeled particle is aluminescent labeled particle containing at least one kind of compoundrepresented by Formula (1) and a particle.

In the present invention, the measurement target substance is measuredby the measurement target substance-related label informationacquisition step of acquiring label information related to the amount ofthe measurement target substance.

The measurement in the present invention is interpreted as the broadestconcept as long as the measurement is measurement of the amount of themeasurement target substance. As a specific embodiment of themeasurement method, a competition method and a sandwich method arementioned, and the competition method is preferable.

As an example of the competition method, a case of quantifyingprogesterone is described below. The same can also be applied to a caseof quantifying substances other than progesterone.

In the competition method, first, a progesterone immunoassay substrateon which a progesterone-albumin conjugate is immobilized is brought intocontact with a biological sample containing progesterone and ananti-progesterone antibody-labeled fluorescent particle. In the casewhere progesterone is not present in the biological sample, anantigen-antibody reaction occurs on the substrate by theanti-progesterone antibody-labeled fluorescent particle and progesteroneon the substrate (that is, progesterone in a progesterone-albuminconjugate). On the other hand, in the case where progesterone is presentin the biological sample, an antigen-antibody reaction occurs betweenprogesterone in the biological sample and the anti-progesteroneantibody-labeled fluorescent particle, and an antigen-antibody reactionbetween the anti-progesterone antibody-labeled fluorescent particle andthe progesterone on the substrate (that is, progesterone in theprogesterone-albumin conjugate) is inhibited. After the above reactionis completed, anti-progesterone antibody-labeled fluorescent particlesthat do not bind to albumin on the substrate are removed. Then, bydetecting a degree of formation of an immune complex (that is, thecomplex of the anti-progesterone antibody-labeled fluorescent particleand progesterone in the progesterone-albumin conjugate on the substrate)on the substrate as fluorescence intensity, the concentration ofprogesterone or the like in the biological sample can be measured.

The measurement form of the fluorescence in the competition method canadopt either plate reader measurement or flow measurement, and forexample, measurement can be performed by the following method. Inadvance, a plurality of samples with known amounts of progesteronehaving different progesterone concentrations are prepared, and thesesamples and the anti-progesterone antibody-labeled fluorescent particlesare mixed in advance. This liquid mixture is brought into contact withan area where the progesterone-albumin conjugate is immobilized. Thefluorescence signal from the area where the progesterone-albuminconjugate is immobilized is measured as a plurality of fluorescencesignals while the liquid mixture is in contact with the conjugate atspecific time intervals. From the plurality of fluorescence signals,temporal change (slope) in the fluorescence amount is acquired at eachprogesterone concentration. The temporal change is plotted as a Y axisand the progesterone concentration is plotted as an X axis, and arelational expression of the progesterone concentration with respect tothe temporal change in the fluorescence amount is acquired using anappropriate fitting method such as the least squares method. The amountof progesterone contained in the biological sample can be quantifiedusing the result of the temporal change in the fluorescence amount usingthe biological sample to be tested based on the relational expressionthus acquired.

It is preferred to perform this quantification of the amount ofprogesterone in a short time. Specifically, the quantification ispreferably performed within 10 minutes, more preferably within 8minutes, and still more preferably within 6 minutes. This quantificationtime preferably includes time required to convert the amount ofprogesterone which is contained in the biological sample, based on theresult of the temporal change in the fluorescence amount acquired usingthe biological sample to be tested after the sample and theanti-progesterone antibody-labeled fluorescent particles are broughtinto contact with detection area where the progesterone-albuminconjugate is immobilized, by using the relational expression between thetemporal change in the fluorescence amount and the progesteroneconcentration, which is acquired in advance using an appropriate fittingmethod such as the least squares method.

The sandwich method is not particularly limited and for example, themeasurement target substance can be measured by the following procedure.A biological sample which may contain a measurement target substance andfluorescent particles having a first binding substance capable ofbinding to the measurement target substance are brought into contactwith each other on a substrate. In the case where the measurement targetsubstance is present in the biological sample, a binding reaction (suchas an antigen-antibody reaction) occurs among the measurement targetsubstance, the fluorescent particles, and the substrate. As a result, inthe case where the measurement target substance is present in thebiological sample, an immune complex including a second bindingsubstance bound to the substrate, the measurement target substance, andthe fluorescent particles having the first binding substance is formed.In the sandwich method, after a reaction among the second bindingsubstance, the measurement target substance, and the fluorescentparticles having the first binding substance is completed, fluorescentparticles having a first binding substance, which do not form theabove-mentioned immune complex, are removed and washing is performed.Next, the concentration of the measurement target substance or the likecan be measured by detecting the degree of the formation of the immunecomplex as fluorescence intensity. The fluorescence intensity and theconcentration of the measurement target substance have a positivecorrelation.

(Flow Channel)

In a preferred aspect of the present invention, a liquid mixtureobtained by mixing a biological sample that may contain a measurementtarget substance and labeled particles having a first binding substanceis applied onto a substrate and developed into a flow channel. The flowchannel is not particularly limited as long as the flow channel is apassage that allows the biological sample and the labeled particleshaving the first binding substance to flow down to the detection area. Apreferred aspect of the flow channel is a flow channel having astructure in which a spotting port for spotting a biological sampleliquid containing the labeled particles having the first bindingsubstance, a metal film as a detection area, and a flow channel beyondthe metal film are provided and the biological sample can pass over themetal film. Preferably, a suction port can be provided on a sideopposite to the spotting port with respect to the metal film.

(Surface Plasmon Fluorescence Measurement)

The method for detecting a label such as fluorescence in the presentinvention is not particularly limited. For example, it is preferred thatfluorescence intensity is detected using a device capable of detectingfluorescence intensity, specifically, a microplate reader or a biosensorfor performing fluorescence detection by surface plasmon excitation(SPF). Preferably, label information related to the amount of themeasurement target substance can be acquired by fluorescence detectionby using surface plasmon resonance.

A form of measurement of fluorescence may be plate reader measurement orflow measurement. In a fluorescence detection method by surface plasmonexcitation (SPF method), the measurement can be performed with highersensitivity than in a fluorescence detection method by epi-excitation(epi-fluorescence method).

As a surface plasmon fluorescence (SPF) biosensor, a sensor described inJP2008-249361A, comprising: an optical waveguide formed of a materialwhich transmits excitation light of a predetermined wavelength; a metalfilm formed on one surface of the optical waveguide; a light source forgenerating a light beam; an optical system for passing the light beamthrough the optical waveguide and causing the light beam to be incidenton an interface between the optical waveguide and the metal film at anincidence angle generating the surface plasmon; and fluorescencedetection means for detecting fluorescence generated by being excited byan evanescent wave enhanced due to the surface plasmon can be used.

The fluorescence detection (SPF) system by surface plasmon excitationusing the fluorescent particles of the present invention is preferablyan assay method for detecting fluorescence from the fluorescentsubstance depending on the amount of the measurement target substanceimmobilized on the metal film on the substrate, and for example, is amethod different from a so-called latex agglutination method in which achange in optical transparency by the progress of a reaction in asolution is detected as turbidity. In the latex agglutination method, anantibody-sensitized latex in a latex reagent and an antigen in abiological sample are bound to be agglutinated by an antibody reaction.The latex agglutination method is a method in which the agglutinateincreases over time, and the antigen concentration is quantified fromthe change in absorbance per unit time obtained by irradiating theagglutinate with near-infrared light. In the present invention, it ispossible to provide a substantially simple method for detecting ameasurement target substance, as compared with the latex agglutinationmethod.

(Standardization)

Furthermore, the method according to the embodiment of the presentinvention may be a method including: a labeled particle-related labelinformation acquisition step of acquiring label information related tothe amount of the labeled particle; and a standardization step ofstandardizing label information acquired in a measurement targetsubstance-related label information acquisition step of acquiring labelinformation related to the amount of the measurement target substance,by the label information acquired in the labeled particle-related labelinformation acquisition step.

In a step of bringing a liquid mixture containing a biological sampleand a labeled particle having a first binding substance capable ofbinding to the measurement target substance into contact with asubstrate having a detection area (test area) and a reference area(control area) to generate the surface plasmon on the detection area andthe reference area, and measuring intensity of emitted fluorescence, astep of measuring intensity of the fluorescence by the surface plasmongenerated on the detection area is the measurement targetsubstance-related label information acquisition step of acquiring labelinformation related to the amount of the measurement target substance,and a step of measuring intensity of the fluorescence by the surfaceplasmon generated on the reference area is the labeled particle-relatedlabel information acquisition step. A step of acquiring an increase ratein the unit time of the fluorescence intensity acquired in these twosteps as change rate of fluorescence signal values and dividing a changerate of signal values of the detection area by a change rate of thesignal value of the reference area is a standardization step.

Hereinafter, the present invention will be described in more detail withreference to the Examples of the present invention. The materials,amounts of use, proportions, treatment contents, treatment procedures,and the like shown in the following Examples can be appropriatelymodified without departing from the spirit and scope of the presentinvention. Therefore, the scope of the present invention should not beinterpreted restrictively by the following specific examples.

EXAMPLES

<1-1> Synthesis of Compound

The terms have the following meanings.

MS: mass spectrometry

ESI: electrospray ionization

NMR: nuclear magnetic resonance

Me: methyl group

Et: ethyl group

Bu: n-butyl group

PL: photoluminescence

THF: tetrahydrofuran

The structures of Compounds (1) to (12) are shown below.

<Synthesis of Compound (1)>

Synthesis of Compound (1-A)

1.00 g of 3,5-bis(trifluoromethyl)benzaldehyde and 20 mL ofdichloromethane were introduced into a 100 mL three-neck flask under anitrogen atmosphere, followed by stirring at room temperature. Whilecooling with water, 0.98 g of 3-ethyl-2,4-dimethylpyrrole was addeddropwise, followed by addition of two drops of trifluoroacetic acid andthen stirring at room temperature for 30 minutes. 1.0 g of chloranil wasadded while cooling with water, followed by stirring at room temperaturefor 10 minutes, and then 3.67 g of diisopropylethylamine (N^(i)Pr₂Et)was added dropwise while cooling with water, followed by stirring atroom temperature for 15 minutes. Subsequently, 5.6 mL of a borontrifluoride-diethyl ether complex was added dropwise while cooling withwater, followed by stirring at room temperature for 30 minutes.Saturated sodium hydrogen carbonate and toluene were added dropwise, andan organic layer obtained by extraction and liquid separation waspreliminarily dried over anhydrous sodium sulfate and then concentratedunder reduced pressure. The resulting crude product was purified bysilica gel column chromatography (developing solvent: hexane/ethylacetate) and then recrystallized from methanol to obtain 1.28 g ofCompound (1-A).

¹H NMR (CDCl₃, 400 MHz): δ 8.03 (s, 1H), 7.83 (s, 2H), 2.54 (s, 6H),2.31 (q, J=7.6 Hz, 4H), 1.21 (s, 6H), 1.00 (t, J=7.6 Hz, 6H).

Synthesis of Compound (1)

100 mg of Compound (1-A), 115 mg of 2,4,6-trimethylbenzaldehyde, and 5mL of dehydrated toluene were introduced into a 100 mL three-neck flask,followed by stirring at room temperature. 1 mL of piperidine and onepiece of p-toluenesulfonic acid monohydrate (manufactured by Wako PureChemical Industries, Ltd., special grade chemical) were added, followedby stirring for 1 hour while distilling off the solvent at 140° C. Afterallowing to cool, 5 mL of dehydrated toluene was added, followed bystirring for 1 hour while distilling off the solvent at 140° C. Thecrude product obtained by concentrating the reaction liquid underreduced pressure was purified by preparative TLC (developing solvent:hexane/ethyl acetate) and then recrystallized from methanol to obtain 71mg of Compound (1). Identification of the compound was carried out by¹H-NMR and ESI-MS.

¹H NMR (CDCl₃, 400 MHz): δ 8.06 (s, 1H), 7.87 (s, 2H), 7.38 (d, J=17.2Hz, 2H), 7.32 (d, J=17.2 Hz, 2H), 6.93 (s, 4H), 2.63 (q, J=7.6 Hz, 4H),2.44 (s, 12H), 2.30 (s, 6H), 1.27 (s, 6H), 1.17 (t, J=7.6 Hz, 6H).

ESI-MS: [M−H]⁻=775.8

<Synthesis of Compound (3)>

Synthesis of Compound (3-A)

16.22 g of 3,5-bis(trifluoromethyl)benzaldehyde and 200 mL ofdichloromethane were introduced into a 1 L three-neck flask under anitrogen atmosphere, followed by stirring at room temperature. 15.75 gof 2,4-dimethylpyrrole was added dropwise while cooling with water,followed by addition of five drops of trifluoroacetic acid and thenstirring at room temperature for 30 minutes. 19.45 g of chloranil wasadded while cooling with water, followed by stirring at room temperaturefor 30 minutes, and 80 mL of diisopropylethylamine (N^(i)Pr₂Et) wasadded dropwise while cooling with water, followed by stirring at roomtemperature for 30 minutes. Subsequently, 85 mL of a borontrifluoride-diethyl ether complex (BF₃.Et₂O) was added dropwise whilecooling with water, followed by stirring at room temperature for 30minutes. 400 mL of saturated sodium hydrogen carbonate was addeddropwise, and an organic layer obtained by extraction and liquidseparation was preliminarily dried over anhydrous sodium sulfate andthen concentrated under reduced pressure. The resulting crude productwas purified by silica gel column chromatography (developing solvent:hexane/ethyl acetate) and then recrystallized from ethanol to obtain4.40 g of Compound (3-A).

Synthesis of Compound (3-B)

3.05 g of Compound (3-A) and 60 mL of 1,1,1,3,3,3-hexafluoro-2-propanolwere introduced into a 300 mL three-neck flask, followed by stirring atroom temperature. 3.60 g of N-iodosuccinimide was introduced, followedby stirring at room temperature for 1.5 hours. After concentrating thereaction liquid under reduced pressure, 50 mL of an aqueous sodiumthiosulfate solution (10 g of sodium thiosulfate dissolved therein) and100 mL of methylene chloride were added, and an organic layer obtainedby extraction and liquid separation was preliminarily dried overanhydrous sodium sulfate and then concentrated under reduced pressure.The resulting crude product was recrystallized from ethanol to obtain3.90 g of Compound (3-B).

Synthesis of Compound (3-C)

2.2 g of Compound (3-B), 2.6 g of 2,4,6-trimethylbenzaldehyde, and 40 mLof dehydrated toluene were introduced into a 100 mL three-neck flask,followed by stirring at room temperature. 4 mL of piperidine wasintroduced, followed by stirring at 65° C. for 1 hour. The crude productobtained by concentrating the reaction liquid under reduced pressure waspurified by silica gel column chromatography (developing solvent:hexane/ethyl acetate) and then recrystallized from ethanol to obtain 2.4g of Compound (3-C).

Synthesis of Compound (3)

96 mg of Compound (3-C), 64 mg of 2,4,6-trimethylphenylboronic acid, 130mg of cesium fluoride, and 10 mL of methoxycyclopentane were introducedinto a 100 mL three-neck flask, followed by degassing under reducedpressure while stirring at room temperature, and the reaction system wasset to a nitrogen atmosphere. 63 mg of SPhos Pd G3 (manufactured bySigma-Aldrich, Inc.) was added thereto, followed by heating under refluxfor 1 hour. 10 mL of a saturated aqueous ammonium chloride solution and10 mL of ethyl acetate were added, and an organic layer obtained byextraction and liquid separation was preliminarily dried over anhydroussodium sulfate and then concentrated under reduced pressure. Theresulting crude product was purified by preparative TLC (developingsolvent: hexane/ethyl acetate) and then recrystallized from ethanol toobtain 16 mg of Compound (3). Identification of the compound was carriedout by ¹H-NMR and ESI-MS.

¹H NMR (CDCl₃, 400 MHz): δ 8.02 (s, 1H), 8.00 (s, 2H), 7.42 (d, J=22.4Hz, 2H), 6.92 (s, 4H), 6.80 (s, 4H), 6.67 (d, J=22.4 Hz, 2H), 2.27 (s,6H), 2.17 (s, 6H), 2.16 (s, 6H), 2.11 (s, 12H), 2.01 (s, 12H).

ESI-MS: [M−H]⁻=955.8

<Synthesis of Compound (2)>

The synthesis was carried out in the same manner as in the synthesis ofCompound (3), except that 3,5-bis(trifluoromethyl)benzaldehyde wasreplaced by 2,3,4,5,6-pentafluorobenzaldehyde and 2,4-dimethylpyrrolewas replaced by 2,4-dimethyl-3-ethylpyrrole. The resulting crude productwas purified by silica gel column chromatography (developing solvent:hexane/ethyl acetate) and then recrystallized fromdichloromethane/methanol to obtain 8 mg of Compound (2). Identificationof the compound was carried out by ¹H-NMR measurement, thus confirmingthe same NMR spectrum as in Org. Biomol. Chem., 2010, Vol. 8, pp. 4546to 4553.

<Synthesis of Compound (4)>

Compound (4) was synthesized in the same manner as in the synthesis ofCompound (2), except that 2,4,6-trimethylbenzaldehyde was replaced byo-tolualdehyde. Identification of the compound was carried out by ¹H-NMRand ESI-MS. A 400 MHz ¹H-NMR spectrum is shown in FIG. 1.

ESI-MS: [M−H]⁻=673.3

<Synthesis of Compound (5)>

Synthesis of Compound (5-A)

1.16 ml of 2,4-dimethylpyrrole and 140 mL of dichloromethane wereintroduced into a 500 mL three-neck flask under a nitrogen atmosphere,followed by stirring at room temperature. 1.0 g of2,3,5,6-tetrafluorobenzaldehyde and one drop of trifluoroacetic acidwere added, followed by stirring at room temperature for 15 minutes.1.38 g of chloranil was added, followed by stirring at room temperaturefor 15 minutes, and then 6.8 mL of diisopropylethylamine (N^(i)Pr₂Et)was added dropwise while cooling with water, followed by stirring atroom temperature for 20 minutes. Subsequently, 7.8 mL of a borontrifluoride-diethyl ether complex (BF₃.Et₂O) was added dropwise whilecooling with water, followed by stirring at room temperature for 30minutes. 400 mL of saturated sodium hydrogen carbonate was addeddropwise, and an organic layer obtained by dichloromethane extractionand liquid separation was preliminarily dried over anhydrous sodiumsulfate and then concentrated under reduced pressure. The resultingcrude product was purified by silica gel column chromatography(developing solvent: hexane/ethyl acetate) and then recrystallized frommethanol to obtain 360 mg of Compound (5-A).

Synthesis of Compound (5-B)

300 mg of Compound (5-A) and 8 mL of 1,1,1,3,3,3-hexafluoro-2-propanolwere introduced into a 300 mL three-neck flask, followed by stirring atroom temperature. 409 mg of N-iodosuccinimide was introduced, followedby stirring at room temperature for 1.5 hours. After concentrating thereaction liquid under reduced pressure, 40 mL of methylene chloride wasadded, and an organic layer obtained by extraction and liquid separationwas preliminarily dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. Ethanol was added to the resultingcrude product, followed by dispersion, washing, and filtration to obtain382 mg of Compound (5-B).

Synthesis of Compound (5-C)

278 mg of Compound (5-B), 564 mg of 2,4,6-trimethylphenylboronic acid,653 mg of cesium fluoride, and 43 mL of methoxycyclopentane wereintroduced into a 100 mL three-neck flask, followed by degassing underreduced pressure while stirring at room temperature, and the reactionsystem was set to a nitrogen atmosphere. 269 mg of SPhos Pd G3(manufactured by Sigma-Aldrich, Inc.) was added thereto, followed byheating under reflux for 1 hour. 250 mL of ethyl acetate was added, andan organic layer obtained by extraction and liquid separation waspreliminarily dried over anhydrous sodium sulfate and then concentratedunder reduced pressure. The resulting crude product was purified bysilica gel column chromatography (developing solvent: hexane/ethylacetate) and then dissolved in 5 ml of dichloromethane, 15 ml ofmethanol was further added, and then dichloromethane was distilled off,followed by reprecipitation. The precipitate was filtered to obtain 206mg of Compound (5-C).

Synthesis of Compound (5)

50 mg of Compound (5-C), 5 ml of toluene, 46 μl of2,4,6-trimethylbenzaldehyde, 400 μl of piperidine, and one piece ofp-toluenesulfonic acid were introduced into a 100 mL three-neck flask,followed by heating under reflux under nitrogen for 1 hour. Afterfurther adding 46 μl of 2,4,6-trimethylbenzaldehyde, followed by heatingunder reflux for 1 hour, 200 l of piperidine was further added, followedby heating under reflux for another 1 hour. After completion of thereaction, the reaction liquid was concentrated under reduced pressure.The resulting crude product was purified by silica gel columnchromatography (developing solvent: hexane/toluene) and then dissolvedin 3 ml of dichloromethane, 15 ml of methanol was added, and thendichloromethane was distilled off, followed by reprecipitation to obtain16 mg of Compound (5). Identification of the compound was carried out by¹H-NMR and ESI-MS.

¹H NMR (CDCl₃, 400 MHz): δ 7.43 (s, 1H), 7.39 (s, 1H), 7.29-7.21 (m,1H), 6.94 (s, 4H), 6.80 (s, 4H), 6.69 (s, 1H), 6.65 (s, 1H), 2.29 (s,6H), 2.23 (s, 6H), 2.08 (s, 12H), 2.03 (s, 12H), 1.33 (s, 6H).

ESI-MS: [M−H]⁻=891.4

<Synthesis of Compound (6)>

Synthesis of Compound (6-A)

1.00 g of 2,3,5,6-tetrafluorobenzaldehyde and 20 mL of dichloromethanewere introduced into a 100 mL three-neck flask under a nitrogenatmosphere, followed by stirring at room temperature. 0.98 g of3-ethyl-2,4-dimethylpyrrole was added dropwise while cooling with water,followed by addition of two drops of trifluoroacetic acid and thenstirring at room temperature for 15 minutes. 1.0 g of chloranil wasadded while cooling with water, followed by stirring at room temperaturefor 10 minutes, and 3.67 g of diisopropylethylamine was added dropwisewhile cooling with water, followed by stirring at room temperature for15 minutes. Subsequently, 5.6 mL of a boron trifluoride-diethyl ethercomplex was added dropwise while cooling with water, followed bystirring at room temperature for 60 minutes. Saturated sodium hydrogencarbonate and toluene were added dropwise, and an organic layer obtainedby extraction and liquid separation was preliminarily dried overanhydrous sodium sulfate and then concentrated under reduced pressure.The resulting crude product was purified by silica gel columnchromatography (developing solvent: toluene) and then recrystallizedfrom methanol to obtain 0.76 g of Compound (6-A).

¹H NMR (CDCl₃, 400 MHz): δ 7.20-7.30 (m, 1H), 2.54 (s, 6H), 2.33 (q,J=7.6 Hz, 4H), 1.51 (s, 6H), 1.01 (t, J=7.6 Hz, 6H).

Synthesis of Compound (6)

181 mg of Compound (6-A), 237 mg of 2,4,6-trimethylbenzaldehyde, and 10mL of dehydrated toluene were introduced into a 100 mL three-neck flask,followed by stirring at room temperature. 2 mL of piperidine and twopieces of p-toluenesulfonic acid monohydrate (manufactured by Wako PureChemical Industries, Ltd., special grade chemical) were added, followedby stirring for 1 hour while distilling off the solvent at 140° C. Thecrude product obtained by concentrating the reaction liquid underreduced pressure was purified by silica gel column chromatography(developing solvent: toluene) and then recrystallized from acetonitrileto obtain 194 mg of Compound (6). Identification of the compound wascarried out by ¹H-NMR and ESI-MS.

¹H NMR (CDCl₃, 400 MHz): δ 7.40 (d, J=17.2 Hz, 2H), 7.32 (d, J=17.2 Hz,2H), 7.20-7.30 (m, 1H), 6.93 (s, 4H), 2.66 (q, J=7.6 Hz, 4H), 2.44 (s,12H), 2.30 (s, 6H), 1.55 (s, 6H), 1.19 (t, J=7.6 Hz, 6H).

ESI-MS: [M−H]⁻=711.7

<Synthesis of Compound (7)>

Compound (7) was synthesized in the same manner as in the synthesis ofCompound (2), except that 2,4,6-trimethylbenzaldehyde was replaced by2,4,6-trimethoxybenzaldehyde. Identification of the compound was carriedout by ¹H-NMR and ESI-MS. A 400 MHz ¹H-NMR spectrum is shown in FIG. 2.

ESI-MS: [M+H]⁺=825.3

<Synthesis of Compound (8)>

Synthesis of Compound (8)

97 mg of Compound (3-C), 58 mg of 2-ethynyl-1,3,5-trimethylbenzene, 3.8mg of copper(I) iodide, 4 mL of THF, and 1 mL of triethylamine wereintroduced into a 50 mL two-neck flask, followed by degassing underreduced pressure while stirring at room temperature, and the reactionsystem was set to a nitrogen atmosphere.Tetrakis(triphenylphosphine)palladium (0) (Pd(PPh₃)₄) was added thereto,followed by heating under reflux for 2 hours. The solvent was removed bydistillation under reduced pressure, and 30 mL of dichloromethane wasadded thereto, followed by washing with 20 mL of water and 20 mL of asaturated aqueous sodium chloride solution. An organic layer waspreliminarily dried over anhydrous sodium sulfate and concentrated underreduced pressure. The resulting crude product was purified by silica gelcolumn chromatography (developing solvent: hexane/toluene) and thenrecrystallized from methanol to obtain 26 mg of Compound (8).Identification of the compound was carried out by ¹H-NMR and ESI-MS.

¹H NMR (CDCl₃, 400 MHz): δ 8.60 (s, 1H), 8.56 (s, 1H), 8.09 (s, 1H),7.90 (s, 2H), 7.41 (s, 1H), 7.37 (s, 1H), 6.88 (s, 4H), 6.85 (s, 4H),2.36 (s, 12H), 2.34 (s, 12H), 2.28 (s, 6H), 2.27 (s, 6H).

ESI-MS: [M−H]⁻=1003.5

<Synthesis of Compound (9)>

Compound (9) was synthesized in the same manner as in the method forsynthesizing Compound (5) through Compounds (5-A) to (5-C), except that2,4,6-trimethylbenzaldehyde in the synthesis of Compound (5) wasreplaced by benzaldehyde.

<Synthesis of Compound (10)>

Compound (10) was synthesized in the same manner as in the method forsynthesizing Compound (5) through Compounds (5-A) to (5-C), except that2,3,5,6-tetrafluorobenzaldehyde in the synthesis of Compound (5-A) wasreplaced by 2,4,6-trimethylbenzaldehyde.

<Synthesis of Compound (11)>

Compound (11) was synthesized in the same manner as in the method forsynthesizing Compound (5) through Compounds (5-A) to (5-C), except that2,4,6-trimethylbenzaldehyde in the synthesis of Compound (5) wasreplaced by 2-formylnaphthalene.

<Synthesis of Compound (12)>

Compound (12) was synthesized in the same manner as in the method forsynthesizing Compound (5) through Compounds (5-A) to (5-C), except that2,4,6-trimethylbenzaldehyde in the synthesis of Compound (5) wasreplaced by 2,6-dimethoxybenzaldehyde.

<1-2> Preparation of High Luminescent Fluorescent Latex DispersionLiquid

Fluorescent latex particles were prepared. As the latex particles,particles having an average particle diameter of 150 nm prepared bypolymerization in a state that a 9/1 (mass ratio) mixture of styrene andacrylic acid was dispersed in water were used. The average particlediameter was measured by a dynamic light scattering method. THF (5 mL)was added dropwise to the above-prepared latex particle dispersionliquid having a solid content of 2% (25 mL of latex dispersion, 500 mgof solid), followed by stirring for 10 minutes. A THF solution (2.5 mL)containing 24 μmol/g of Compound (1) was added thereto dropwise over 15minutes. Regarding the amount of compound in a table, μmol/g representsthe number of moles of the compound used per 1 g of solids in the latex,and % by mass represents the % by mass of the compound used per 1 g ofsolids in the latex. Completion of the dropwise addition of the compoundwas followed by stirring for 30 minutes and concentrating under reducedpressure to remove THF. Thereafter, the particles were precipitated bycentrifugation, followed by addition of ultrapure water, and thendispersed again to produce a high luminescent fluorescent latexdispersion liquid having a solid content concentration of 2%.

Further, the same operation was performed using Compound (5) instead ofCompound (1) to produce a high luminescent fluorescent latex dispersionliquid having a solid content concentration of 2% containing Compound(5).

Furthermore, the same operation was performed using a mixture of 24μmol/g of Compound (1) and 12 μmol/g of Compound (5) to produce a highluminescent fluorescent latex dispersion liquid having a solid contentconcentration of 2% containing Compound (1) and Compound (5).

<2-1> Preparation of Fluorescent Particles Labeled withAnti-Progesterone Antibody

High luminescent fluorescent latex particles (average particle diameterof 150 nm) labeled with anti-progesterone antibody were prepared asfollows.

117 μL of a buffer solution (pH of 6.0) of 50 mM2-morpholinoethanesulfonic acid (MES, manufactured by Dojindo MolecularTechnologies, Inc.) and 5 μL of an aqueous solution of 10 mg/mLwater-soluble carbodiimide (WSC) were added to 375 μL of a highluminescent fluorescent latex dispersion liquid having a solid contentconcentration of 2% by mass prepared in <1-2>, followed by stirring atroom temperature for 15 minutes. Subsequently, 182.4 μL of a 0.5 mg/mLanti-progesterone monoclonal antibody (manufactured by GeneTex, Inc.)was added, followed by stirring at room temperature for 1.5 hours. 37.5μL of an aqueous solution of 2 mol/L glycine (manufactured by Wako PureChemical Industries, Ltd.) was added, followed by stirring for 15minutes, and then the fluorescent latex particles were precipitated bycentrifugation (15,000 rpm, 4° C., 30 minutes). A supernatant liquid wasremoved, 750 μL of a phosphate buffered saline (PBS, manufactured byWako Pure Chemical Industries, Ltd.) solution (pH of 7.4) was added, andthe fluorescent latex particles were re-dispersed with an ultrasoniccleaner. Centrifugation (15,000 rpm, 4° C., 15 minutes) was performed, asupernatant liquid was removed, followed by addition of 750 μL of a PBS(pH of 7.4) solution containing 1% by mass of BSA, and then thefluorescent latex particles were re-dispersed to obtain a solution of 1mass % anti-progesterone antibody-bound fluorescent latex particles.

<2-2> Preparation of Solution of Progesterone-BSA Conjugate in CitrateBuffer Solution

150 μg of a progesterone-BSA conjugate (manufactured by Bio-RadLaboratories, Inc.) was added to and dissolved in 1 mL of a citratebuffer solution at a concentration of 50 mM (pH of 5.2, 150 mM NaCl),thereby obtaining a solution of a citrate buffer solution.

<2-3> Production of Progesterone-BSA Conjugate-Immobilized Substrate

A polymethyl methacrylate (PMMA) substrate (manufactured by MitsubishiRayon Co., Ltd., ACRYPET (registered trademark) VH) was prepared, a goldfilm having thickness of 50 nm was deposited on one side thereof by avapor deposition method, and the resultant is cut to a width of 7 mm isperformed to prepare seven identical substrates. The solution of theprogesterone-BSA conjugate in the citrate buffer solution prepared in 1.was spotted on a gold-deposited surface of the substrate, and dried toprepare a substrate on which the progesterone-BSA conjugate isimmobilized.

<2-4> Washing and Blocking of Substrate

Before the substrate prepared in this manner is attached to a flowchannel of a sensor chip, a PBS solution (pH of 7.4) containing Tween 20(polyoxyethylene (20) sorbitan monolaurate, Wako Pure ChemicalIndustries, Ltd.) at a concentration of 0.05% by mass was prepared inadvance, and the substrate was repeatedly washed three times with 300 μLof the solution. After completion of the washing, in order to block aportion which was not adsorbed with the T4-BSA conjugate, on thegold-deposited film, 300 μL of a PBS solution (pH of 7.4) containing 1%by mass of casein (manufactured by Thermo Fisher Scientific Inc.) wasadded, followed by being left to stand for 1 hour at room temperature.After washing with the solution for washing, 300 μL of ImmunoassayStabilizer (manufactured by Advanced Biotechnologies Inc.) was added asa stabilizer, followed by being left to stand for 30 minutes at roomtemperature. Then, the solution was removed and moisture was completelyremoved using a dryer.

<2-5> Preparation of Sensor Chip

A flow channel-type sensor chip was prepared to have the configurationof the second embodiment in JP2010-190880A. The schematic views thereofare shown in FIG. 3 and FIG. 4. FIG. 3 is a schematic view of a sensorchip 1, and FIG. 4 is an exploded view of the sensor chip 1. The sensorchip 1 includes an upper member 2, an intermediate member 3, and asubstrate 4. The upper member 2 is provided with a first container 5 anda second container 6. The first container 5 and the second container 6are collectively referred to as a container group 7. A flow channel 10is formed in the substrate 4, and a detection area 8 and a referencearea 9 are formed on the flow channel 10.

<3-1> Measurement with Large-Scale Apparatus (Pre-Existing ProgesteroneMeasurement Reagent)

In immunoassay, measurement of a sample in which an amount ofprogesterone is known was performed by using IMMULYZE 1000 (LSI MedienceCorporation), which is a large-scale apparatus widely used by thoseskilled in the art and according to the instruction manual, and measuredvalues of progesterone were obtained.

<3-2> Measurement of Progesterone Using Antibody-Labeled Particles

Samples containing progesterone having various concentrations (0.00ng/mL, 0.5 ng/mL, 2.0 ng/mL, 15.0 ng/mL, 30.0 ng/mL, and 45.0 ng/mL)were prepared. Next, a plurality of cups for antibody-labeled particleconcentrations were prepared so that concentrations of antibody-labeledparticles became 0.080%, 0.040%, 0.020%, 0.010%, 0.005%, 0.002%, and0.001% at a time of preparing liquid mixtures of the antibody-labeledparticles labeled with anti-progesterone antibody prepared in <2-1> andthe various samples, the samples having various concentrations preparedabove were added to the cups for respective concentrations, and whilestirring for 10 minutes, the liquid mixtures of the antibody-labeledparticles and the samples were prepared. Then, on the flow channel-typesensor chip in which the substrate was sealed, prepared in 2-5, theliquid mixtures of the antibody-labeled particles and the samples wererespectively spotted. After spotting was completed, the liquid mixtureswere allowed to flow down at a rate of 10 μL/min while pump suction wasperformed, and brought into contact with the surface of the gold film onwhich the progesterone-BSA conjugate was immobilized, and then themeasurement of fluorescence intensity was continued for 1.5 minutes.Standardization was performed by acquiring an increase rate in the unittime of the fluorescence intensity of each of the detection area and thereference area obtained in each substrate, as a fluorescence signalvalue, and dividing the signal value of the detection area by the signalvalue of the reference area. In addition, a sample with a progesteroneconcentration of zero was prepared, and in the same manner as describedabove, standardization of a signal value from a sample withoutprogesterone was performed.

<4> Creation of Calibration Curve

By acquiring the correlation between the fluorescence signal valuestandardized from the sample with the known amount of progesteroneobtained in <3-2> and the value measured with the large-scale apparatusacquired in <3-1>, a calibration curve was created for the substrateusing the progesterone-BSA conjugate prepared in <2-2>. Literature “TheImmunoassay Handbook Third Edition Edited by David Wild (2005)”describes that a four-parameter logistic curve model of a sigmoidfunction can be applied as a calibration curve of a competition method,and according to this method, a four-parameter logistic curve passingthe nearest neighbor of each point of the fluorescence signal values atthe respective progesterone concentrations measured in 3-2. was acquiredusing the least squares method generally known as a method for obtainingan approximate line, and the curve was set as a calibration curve.

From the calibration curve acquired as described above, the measuredvalue of the sample with each progesterone concentration was calculated.

The performance was determined according to whether to satisfy thestandard of the calibration curve. The calibration curve was defined bytwo points. A first point was a slope of the calibration curve in a lowconcentration range, and a case where a reciprocal of the slope iswithin 2.0 was set as a standard. A second point was a deviation fromthe calibration curve at the measurement point in a high concentrationrange, and a case where the deviation was within 4% was set as astandard. Within this range, it is possible to achieve the coefficientof variation of the measured value within 10% and the accuracy within10%, and to perform the high-precision measurement.

In the case of the low concentration range, a slope of a calibrationcurve at 0.5 ng/mL as the minimum concentration of progesterone which isclinically meaningful was acquired. In addition, in the case of the highconcentration range, deviations from respective calibration curves atprogesterone concentrations of 30.0 ng/mL and 45.0 ng/mL were acquired,and an average value thereof was calculated and evaluated. The resultsare summarized in Table 3.

<5> Preparation of Comparative Particles

<5-1> Preparation of Comparative Fluorescent Latex Particle DispersionLiquid

100 mL of methanol was added to 100 mL of an aqueous dispersion liquidhaving the solid content concentration of the latex particle dispersionliquid prepared in <1-2> of 2% by mass, followed by stirring at roomtemperature for 10 minutes. On the other hand, a separately preparedfluorescent dye (comparative compound: Compound 5 described inJP3442777B) solution (dissolved in 1 mL of N,N-dimethylformamide, 9 mLof CHCl₃, and 16 mL of ethanol) was gradually added dropwise into thelatex particle dispersion liquid over 60 minutes. After completion ofthe dropwise addition, an organic solvent was distilled off underreduced pressure with an evaporator, and then centrifugation andredispersion in an aqueous PBS solution were repeated three times toperform purification, thereby preparing a comparative fluorescent latexparticle dispersion liquid.

<5-2> Preparation of Comparative Antibody-Labeled Particles Labeled withAnti-Progesterone Antibody

In the preparation of the fluorescent latex particles prepared in <2-1>,the fluorescent latex particles were replaced by the comparativefluorescent latex particles of 2% by mass (solid content concentration)prepared in <5-1>, and other operations were performed in the samemanner to prepare comparative antibody-labeled particles. The otheroperations were carried out in the same manner to the measurement of<4>.

<6> Measurement of Particle Fluorescence Intensity

The fluorescence latex dispersion liquid having a solid contentconcentration of 2% by mass was diluted 200 times with ultrapure water,the excitation light of a fluorescence spectrophotometer RF-5300PC(manufactured by Shimadzu Corporation) was set to 658 nm, andmeasurement was performed. In the case where the fluorescence intensityof the fluorescent latex dispersion liquid was high enough to exceed themeasurement range, dilution was performed with ultrapure water to arange in which the maximum value of the fluorescence intensity wasmeasurable. An integrated value of the fluorescence intensity of theemission spectrum of the fluorescent latex dispersion liquid withrespect to an integrated value of the fluorescence intensity of theemission spectrum of the fluorescent latex dispersion liquid prepared in7-1. was taken as the particle fluorescence intensity (relative value).A calculation expression used for the calculation is shown below.

Fluorescence intensity (relative value)=(Integrated value offluorescence intensity of emission spectrum of fluorescent latexdispersion liquid)/(Integrated value of fluorescence intensity ofemission spectrum of fluorescent latex dispersion liquid prepared inComparative example)

<Evaluation Standards>

The determination was set as A in the case where the reciprocal of theslope of the calibration curve in the low concentration range was 2.0 orless, and the determination was set as B in the case where thereciprocal thereof was larger than 2.0.

The determination was set as A in the case where the deviation from thecalibration curve in the high concentration range was 4% or less, andthe determination was set as B in the case where the deviation therefromwas larger than 4%.

TABLE 3 Reciprocal of slope of Deviation from calibration calibrationParticle curve in low curve in high fluorescence concentrationconcentration Particle intensity range range Compound concentration(relative value) Determination Determination Example 1 (1), (5) 0.080%by mass 12.1 1.7 A 1.8% A Example 2 (1), (5) 0.040% by mass 12.1 1.6 A1.9% A Example 3 (1), (5) 0.020% by mass 12.1 1.5 A 2.0% A Example 4(1), (5) 0.010% by mass 12.1 1.4 A 2.4% A Example 5 (1), (5) 0.005% bymass 12.1 1.3 A 2.9% A Example 6 (1), (5) 0.002% by mass 12.1 1.2 A 3.3%A Example 7 (1), (5) 0.001% by mass 12.1 1.1 A 3.4% A Example 8 (1)0.080% by mass 5.8 2.0 A 2.0% A Example 9 (1) 0.040% by mass 5.8 1.9 A2.4% A Example 10 (1) 0.020% by mass 5.8 1.8 A 2.8% A Example 11 (1)0.010% by mass 5.8 1.7 A 3.2% A Example 12 (1) 0.005% by mass 5.8 1.6 A3.4% A Example 13 (1) 0.002% by mass 5.8 1.5 A 3.6% A Example 14 (1)0.001% by mass 5.8 1.4 A 3.8% A Example 15 (5) 0.080% by mass 7.0 1.9 A1.9% A Example 16 (5) 0.040% by mass 7.0 1.8 A 2.2% A Example 17 (5)0.020% by mass 7.0 1.7 A 2.5% A Example 18 (5) 0.010% by mass 7.0 1.6 A3.1% A Example 19 (5) 0.005% by mass 7.0 1.5 A 3.3% A Example 20 (5)0.002% by mass 7.0 1.4 A 3.5% A Example 21 (5) 0.001% by mass 7.0 1.3 A3.6% A Comparative Comparative 0.080% by mass 1.0 3.8 B 2.0% A Example 1compound Comparative Comparative 0.040% by mass 1.0 2.0 A 4.1% B Example2 compound Comparative Comparative 0.020% by mass 1.0 1.9 A 4.5% BExample 3 compound Comparative Comparative 0.010% by mass 1.0 1.8 A 5.2%B Example 4 compound Comparative Comparative 0.005% by mass 1.0 1.7 A7.2% B Example 5 compound Comparative Comparative 0.002% by mass 1.0 1.6A 9.0% B Example 6 compound Comparative Comparative 0.001% by mass 1.01.5 A 10.2% B Example 7 compound

As shown in the results in Table 3, in the particles of Comparativeexamples having low particle fluorescence intensity, in the case wherethe particle concentration was high, the slope of the calibration curvein the low concentration range could not be obtained and in the casewhere the particle concentration was low, the deviation from thecalibration curve in the high concentration range became large.Therefore, there is no condition in which the measurement with highprecision could be performed over the entire measurement range. Incontrast, it was found that the high luminescent fluorescent particlesof the present invention can be measured with high precision over theentire measurement range, and the effect of the present invention wasconfirmed.

EXPLANATION OF REFERENCES

-   -   1 Sensor chip    -   2 Upper member    -   3 Intermediate member    -   4 Substrate    -   5 First container    -   6 Second container    -   7 Container group    -   8 Detection area    -   9 Reference area    -   10 Flow channel

What is claimed is:
 1. A kit for measuring a measurement targetsubstance in a biological sample, the kit comprising: a labeled particlehaving a first binding substance capable of binding to a measurementtarget substance in a biological sample; and a substrate having a secondbinding substance capable of binding to any one of the measurementtarget substance or the first binding substance, wherein the labeledparticle is a luminescent labeled particle containing at least one kindof compound represented by Formula (1) and a particle,

in the formula, R¹¹ to R¹⁵ each independently represent a hydrogen atom,a halogen atom, an alkyl group, an aryl group, a heterocyclic group, anethenyl group, an ethynyl group, an amino group, an acyl group, analkoxy group, an aryloxy group, an alkylthio group, or an arylthiogroup, each of which may have a substituent, and at least three of R¹¹,. . . , or R¹⁵ represent atoms or groups other than hydrogen atoms; X¹and X² each independently represent a halogen atom, an alkyl group, anaryl group, a heterocyclic group, a hydroxy group, an alkoxy group, anaryloxy group, an alkylthio group, an arylthio group, an ethenyl group,or an ethynyl group, each of which may have a substituent, and X¹ and X²may be linked to each other to form a ring; Ar¹ and Ar² eachindependently represent an aryl group or a heterocyclic group, each ofwhich may have a substituent; and L¹ and L² each independently representany one of Formulae (L-1) to (L-4),

in the formulae, R¹¹¹ to R¹¹⁶ each independently represent a hydrogenatom, a halogen atom, an alkyl group, an aryl group, a heterocyclicgroup, an ethenyl group, an ethynyl group, an amino group, an acylgroup, an alkoxy group, an aryloxy group, an alkylthio group, or anarylthio group, each of which may have a substituent; and A represents—O—, —S—, or —NH—.
 2. The kit according to claim 1, wherein the labeledparticle is a labeled latex particle.
 3. The kit according to claim 1,wherein the labeled particle has a carboxyl group.
 4. The kit accordingto claim 1, wherein an average particle size of the labeled particle is70 to 500 nm.
 5. The kit according to claim 1, wherein the compoundrepresented by Formula (1) is a compound represented by Formula (3),

in the formula, R¹¹, R¹², R¹⁴, R¹⁵, X¹, X², Ar¹, Ar², L¹, and L² are asdefined in Formula (1), provided that at least two of R¹¹, R¹², R¹⁴, orR¹⁵ are atoms or groups other than hydrogen atoms; and R³¹ to R³⁵ eachindependently represent a hydrogen atom, a halogen atom, an alkyl group,an aryl group, a heterocyclic group, an ethenyl group, an ethynyl group,an amino group, an acyl group, a cyano group, an alkoxy group, anaryloxy group, an alkylthio group, or an arylthio group, each of whichmay have a substituent, and any one of R³¹, R³², R³⁴, or R³⁵ is a groupconsisting of two or more atoms.
 6. The kit according to claim 1,wherein the compound represented by Formula (1) is a compoundrepresented by Formula (4),

in the formula, R¹², R¹³, R¹⁴, X¹, X², Ar¹, Ar², L¹, and L² are asdefined in Formula (1), provided that at least one of R¹², R¹³, or R¹⁴is an atom or group other than a hydrogen atom; and R⁴¹ and R⁴² eachindependently represent an aryl group, a heterocyclic group, an ethenylgroup, or an ethynyl group, each of which may have a substituent.
 7. Thekit according to claim 1, wherein the compound represented by Formula(1) is a compound represented by Formula (5),

in the formula, R¹¹ to R¹⁵, X¹, X², L¹, and L² are as defined in Formula(1); R⁵¹ and R⁵² each independently represent an alkyl group, an arylgroup, a heteroaryl group, an amino group, an acyl group, an alkoxygroup, an aryloxy group, an alkylthio group, or an arylthio group, eachof which may have a substituent; and Q¹ and Q² each independentlyrepresent an aromatic hydrocarbon ring or an aromatic heterocyclic ring,each of which may have a substituent.
 8. The kit according to claim 1,wherein the labeled particle is a luminescent particle containing atleast one kind of energy donor compound, at least one kind of energyacceptor compound, and a particle, and at least one kind of the energydonor compound or the energy acceptor compound is the compoundrepresented by Formula (1).
 9. The kit according to claim 8, wherein atleast one kind of compound represented by Formula (1) is contained asthe energy donor compound, and at least one kind of compound representedby Formula (1) is contained as the energy acceptor compound.
 10. The kitaccording to claim 8, wherein a molar ratio of the energy donor compoundto the energy acceptor compound is 1:10 to 10:1.
 11. The kit accordingto claim 8, wherein a Stokes shift between the energy donor compound andthe energy acceptor compound is 40 nm or more.
 12. The kit according toclaim 1, wherein the substrate includes a detection area having thesecond binding substance.
 13. The kit according to claim 12, wherein thedetection area is a metal film containing gold.
 14. A method formeasuring a measurement target substance in a biological sample, themethod comprising: a reaction step of reacting a biological sample witha labeled particle having a first binding substance capable of bindingto a measurement target substance; a capturing step of capturing thelabeled particle on a substrate having a second binding substancecapable of binding to any one of the measurement target substance or thefirst binding substance by bringing a reaction product obtained in thereaction step into contact with the substrate; and a label informationacquisition step of acquiring label information related to themeasurement target substance, wherein the labeled particle is aluminescent labeled particle containing at least one kind of compoundrepresented by Formula (1) and a particle,

in the formula, R¹¹ to R¹⁵ each independently represent a hydrogen atom,a halogen atom, an alkyl group, an aryl group, a heterocyclic group, anethenyl group, an ethynyl group, an amino group, an acyl group, analkoxy group, an aryloxy group, an alkylthio group, or an arylthiogroup, each of which may have a substituent, and at least three of R¹¹,. . . , or R¹⁵ represent atoms or groups other than hydrogen atoms; X¹and X² each independently represent a halogen atom, an alkyl group, anaryl group, a heterocyclic group, a hydroxy group, an alkoxy group, anaryloxy group, an alkylthio group, an arylthio group, an ethenyl group,or an ethynyl group, each of which may have a substituent, and X¹ and X²may be linked to each other to form a ring; Ar¹ and Ar² eachindependently represent an aryl group or a heterocyclic group, each ofwhich may have a substituent; and L¹ and L² each independently representany one of Formulae (L-1) to (L-4),

in the formulae, R¹¹¹ to R¹¹⁶ each independently represent a hydrogenatom, a halogen atom, an alkyl group, an aryl group, a heterocyclicgroup, an ethenyl group, an ethynyl group, an amino group, an acylgroup, an alkoxy group, an aryloxy group, an alkylthio group, or anarylthio group, each of which may have a substituent; and A represents—O—, —S—, or —NH—.
 15. The method according to claim 14, wherein thelabeled particle is a luminescent particle containing at least one kindof energy donor compound, at least one kind of energy acceptor compound,and a particle, and at least one kind of the energy donor compound orthe energy acceptor compound is the compound represented by Formula (1).16. The method according to claim 15, wherein at least one kind ofcompound represented by Formula (1) is contained as the energy donorcompound, and at least one kind of compound represented by Formula (1)is contained as the energy acceptor compound.
 17. The method accordingto claim 15, wherein a molar ratio of the energy donor compound to theenergy acceptor compound is 1:10 to 10:1.
 18. The method according toclaim 15, wherein a Stokes shift between the energy donor compound andthe energy acceptor compound is 40 nm or more.
 19. The method accordingto claim 14, wherein the substrate includes a detection area having thesecond binding substance.
 20. The method according to claim 14, whereinlabel information related to the measurement target substance isacquired by fluorescence detection due to surface plasmon excitation.